Comestible compositions comprising high potency savory flavorants, and processes for producing them

ABSTRACT

The present invention relates to the use of certain high potency savory (‘umami’) taste modifiers, as savory flavoring agents and/or enhancers of monosodium glutamate, for the preparation of foods, beverages, and other comestible compositions, and to processes for preparing food flavorant compositions for use in the preparation of comestible food and drink.

RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 12/297,986, filed on May 13, 2009, now U.S. Pat.No. 8,148,536, which is a U.S. national stage application ofInternational Application No. PCT/US2007/009828, filed on Apr. 19, 2007,which, in turn, claims the benefit of priority of U.S. provisionalpatent application Ser. No. 60/793,844, filed on Apr. 21, 2006, and alsoclaims the priority of U.S. provisional patent application Ser. No.60/847,632, filed Sep. 27, 2006, the entire disclosures of which arehereby incorporated herein by this reference.

FIELD OF THE INVENTION

The inventions disclosed herein relate to the use of certain highpotency savory (“umami”) taste modifiers, as savory flavoring agentsand/or enhancers of monosodium glutamate, for the preparation of foods,beverages, and other comestible compositions, and to processes forpreparing food flavorant concentrate compositions for use in thepreparation of comestible food and drink. The inventions also relate toprocesses for preparing some of the savory tastant compounds disclosedherein.

BACKGROUND OF THE INVENTION

For centuries, various natural and unnatural compositions and/orcompounds have been added to comestible (edible) foods and beverages toimprove their taste. Although it has long been known that there are onlya few basic types of “tastes,” the biological and biochemical basis oftaste perception was poorly understood, and most taste improving ortaste modifying agents have been discovered largely by simple trial anderror processes.

For example, one of the five known basic tastes is the “savory” or“umami” flavor of monosodium glutamate (“MSG”), synthetic or naturalversions of which are often added to foods, often at concentrations onthe order of about 0.05 to about 0.5% by weight. Alternatively, MSG ispresent in and can be added in the form of certain food additives, suchas autolyzed yeast extracts (“AYE”) or hydrolyzed vegetable proteins(“HVP”), which are often added to comestible foods and drinks at aconcentration from about 0.1 to about 2% by weight. MSG is however knownto produce adverse reactions in some people, and MSG comprisessignificant amounts of undesirable sodium, but very little progress hasbeen made in identifying artificial substitutes for MSG.

It is also known that a few naturally occurring materials can increaseor enhance (multiply) the effectiveness of MSG as a savory flavoringagent, so that less MSG is needed for a given flavoring application. Forexample, the naturally occurring nucleotide

compounds inosine monophosphate (IMP) and guanosine monophosphate (GMP)are known to have a multiplier (“enhancer”) effect on the savory tasteof MSG. IMP and GMP can also be present in AYE or HVP food additives,but are difficult and expensive to isolate and purify from naturalsources, or synthesize, and hence have limited practical applications.High potency compounds that would substitute for the savory flavor ofMSG₅ or enhance the effectiveness of any MSG present, so that less MSGcould be employed in food compositions, could be of very high value.

In recent years substantial progress has been made in biotechnology ingeneral, and in better understanding the underlying biological andbiochemical phenomena of taste perception. For example, taste receptorproteins have been recently identified in mammals which are involved intaste perception. Particularly, two different families of G proteincoupled receptors believed to be involved in taste perception, T2Rs andT1Rs, have been identified. (See, e.g., Nelson, et al., Cell (2001)106(3):381-390; Adler, et al., Cell (2000) 100(6):693-702;Chandrashekar, et al., Cell (2000) 100:703-711; Matsunami, et al.,Number (2000) 404:601-604; Li, et al., Proc. Natl. Acad. Sci. USA (2002)99:4962-4966; Montmayeur, et al., Nature Neuroscience (2001)4(S):492-498: U.S. Pat. No. 6,462,148; and PCT publications WO 02/06254,WO 00/63166 art, WO 02/064631, and WO 03/001876, and U.S. PatentPublication No. US 2003/0232407 A1).

Whereas the T2R family includes a family of over 25 genes that areinvolved in bitter taste perception, the T1Rs only includes threemembers, T1R1, T1R2, and T1R3. (See Li, et al., Proc. Natl. Acad. Sci.USA (2002) 99:4962-4966.) Recently it was disclosed in WO 02/064631and/or WO 03/001876 that certain T1R members, when co-expressed insuitable mammalian cell lines, assemble to form functional tastereceptors. Particularly it was found that co-expression of T1R1 and T1R3in a suitable host cell results in a functional T1R1/T1R3 savory(“umami”) taste receptor that responds to savory taste stimuli,including MSG.

More recently, certain publications disclosed the discovery and use ofcertain amide compounds as very high potency umami tastants and/orsynergistic enhancers of the “Umami” taste of MSG.

Nevertheless, the current applicants have unexpectedly discovered that,like many other known artificial flavorants, at higher concentrationssome of the new high potency compounds can have flavor differences ascompared to MSG, such as mouth-watering side-tastes, a flavor“lingering” as compared to MSG, or in some cases a perception of tonguetingling or numbness. While such side tastes can actually be desirablein some food formulations (e.g., hot and spicy sauces), minimization ormasking of any side tastes can, in other applications, be desirable.Such side tastes can become noticeable if the high potency savorycompounds are not well dispersed within the comestible compositions.Moreover, while the solubility of the newly discovered high potencycompounds is often good in aqueous and polar organic media, solubilitycan be limited in hydrophobic/lipophilic materials such as fats andoils, which are a natural component of many foods. Accordingly,effective formulation of the new high potency savory flavor compounds toachieve optimal human perception of the savory/Umami flavors, whileminimizing side tastes, can sometimes be difficult.

However, when a new chemical entity such as new high potency savorycompounds have been discovered that is safe for human use, the originallaboratory process by which the compound was first made may not beoptimal for the production of commercial quantities. No large scalesynthetic procedures for the preparation of the high potency savorytastant compoundsN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide and2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide have beendisclosed in the prior art, and as such, investigation of suitableprocesses for large scale preparations of the compounds recited abovewas undertaken.

To this end, the preparation of savory modulating and/or enhancingformulations that can provide a perceptible savory flavor and/or enhancethe flavor of MSG, but avoid and/or overcome issues related to sidetastes and solubility and can be used in a variety of applications isneeded. The compositions and savory flavorant concentrate compositionsand methods disclosed herein meet these unexpected complications andcomplexities.

SUMMARY OF THE INVENTION

The inventions described herein have many aspects, some of which relateto processes for formulating comestible compositions comprising highpotency compounds having a savory “Umami” flavor or that can serve asenhancers for the savory “Umami flavor” of monosodium glutamate, and tothe comestible products themselves, or to certain aspects of improvedmethods for making or purifying the compounds themselves.

The foregoing discussion merely summarizes certain aspects of theinventions and is not intended, nor should it be construed, as limitingthe invention in any way.

DETAILED DESCRIPTION OF THE INVENTION

The inventions described herein can be understood more readily byreference to the following detailed description of various aspects ofthe invention and the Examples included therein and to the chemicaldrawings and Tables and their previous and following description. Beforethe present compounds, compositions, and/or methods are disclosed anddescribed, it is to be understood that unless otherwise specificallyindicated by the claims, the inventions are not limited to specificfoods or food preparation methods, specific comestibles carriers orformulations, or to particular modes of formulating the compounds of theinvention into comestible products or compositions intended for oraladministration, because as one of ordinary skill in relevant arts iswell aware, such things can of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only and is not intended to be limiting.

DEFINITIONS

A “comestibly, biologically or medicinally acceptable carrier orexcipient” is a solid or liquid medium and/or composition that is usedto prepare a desired dosage form of the inventive compounds, in order toadminister the inventive compounds in a dispersed/diluted form, so thatthe biological effectiveness of the inventive compounds are maximized. Acomestibly, biologically or medicinally acceptable carrier includes manycommon food ingredients, such as water at neutral, acidic, or basic pH,fruit or vegetable juices, vinegar, marinades, beer, wine, naturalwater/fat emulsions such as milk or condensed milk, edible oils andshortenings, fatty acids, low molecular weight oligomers of propyleneglycol, glyceryl esters of fatty acids, and dispersions or emulsions ofsuch hydrophobic substances in aqueous media, salts such as sodiumchloride, wheat flours, solvents such as ethanol, solid edible diluentssuch as vegetable powders or flours, or other liquid vehicles;dispersion or suspension aids; surface active agents; isotonic agents;thickening or emulsifying agents, preservatives; solid binders;lubricants and the like.

A “flavor” herein refers to the perception of taste and/or smell in asubject, which include sweet, sour, salty, bitter, umami, and others.The subject may be a human or an animal.

A “flavoring agent” herein refers to a compound or a biologicallyacceptable salt thereof that induces a flavor or taste in a animal or ahuman.

A “flavor modifier” herein refers to a compound or biologicallyacceptable salt thereof that modulates, including enhancing orpotentiating, and inducing, the tastes and/or smell of a natural orsynthetic flavoring agent in a animal or a human.

A “flavor enhancer” herein refers to a compound or biologicallyacceptable salt thereof that enhances or “multiplies” the tastes orsmell of a natural or synthetic flavoring agent.

“Savory flavor” herein refers to the savory, “mouth-watering,” “umami”taste sensation typically induced by MSG (monosodium glutamate) in aanimal or a human.

“Savory flavoring agent,” “savory compound,” or “savory receptoractivating compound” herein refers to a compound or biologicallyacceptable salt thereof that elicits a detectable savory flavor in asubject, e.g., MSG (monosodium glutamate) or a compound that activates aT1R1/T1R3 receptor in vitro. The subject may be a human or an animal.

A “savory flavor modifier” herein refers to a compound or biologicallyacceptable salt thereof that modulates, including enhancing orpotentiating, inducing, and blocking, the savory taste of a natural orsynthetic savory flavoring agents, e.g., monosodium glutamate (MSG) in aanimal or a human.

A “savory flavor enhancer” herein refers to a compound or biologicallyacceptable salt thereof that enhances, potentiates, or “multiplies” thesavory taste of a natural or synthetic savory flavoring agents, e.g.,monosodium glutamate (MSG) in a animal or a human.

An “umami receptor activating compound” herein refers to a compound thatactivates an umami receptor, such as a T1R1/T1R3 receptor.

An “umami receptor modulating compound” herein refers to a compound thatmodulates (activates, enhances or blocks) an umami receptor.

An “umami receptor enhancing compound” herein refers to a compound thatenhances or potentiates the effect of a natural or synthetic umamireceptor activating compound, e.g., monosodium glutamate (MSG).

A “savory flavoring agent amount” herein refers to an amount of acompound that is sufficient to induce savory taste in a comestible ormedicinal product or composition, or a precursor thereof. A fairly broadrange of a savory flavoring agent amount can be from about 0.001 ppm to100 ppm, or a narrow range from about 0.1 ppm to about 10 ppm.Alternative ranges of savory flavoring agent amounts can be from about0.01 ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, fromabout 0.1 ppm to about 5 ppm, or from about 0.1 ppm to about 3 ppm.

A “savory flavor modulating amount” herein refers to an amount of acompound of Formula (I) that is sufficient to alter (either increase ordecrease) savory taste in a comestible or medicinal product orcomposition, or a precursor thereof, sufficiently to be perceived by ahuman subject. A fairly broad range of a savory flavor modulating amountcan be from about 0.001 ppm to 100 ppm, or a narrow range from about 0.1ppm to about 10 ppm. Alternative ranges of savory flavor modulatingamounts can be from about 0.01 ppm to about 30 ppm, from about 0.05 ppmto about 15 ppm, from about 0.1 ppm to about 5 ppm, or from about 0.1ppm to about 3 ppm.

A “savory flavor enhancing amount” herein refers to an amount of acompound that is sufficient to enhance the taste of a natural orsynthetic flavoring agents, e.g., mono sodium glutamate (MSG) in acomestible or medicinal product or composition. A fairly broad range ofa savory flavor enhancing amount can be from about 0.001 ppm to 100 ppm,or a narrow range from about 0.1 ppm to about 10 ppm. Alternative rangesof savory flavor enhancing amounts can be from about 0.01 ppm to about30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about5 ppm, or from about 0.1 ppm to about 3 ppm.

An “umami receptor modulating amount” herein refers to an amount of acompound that is sufficient to modulate (activate, enhance or block) anumami receptor. A preferable range of an umami receptor modulatingamount is 1 μM to 100 mM and most preferably 1 nM to 100 μM and mostpreferably 1 nM to 30 μM. A fairly broad range of an umami flavorenhancing amount can be from about 0.001 ppm to 100 ppm, or a narrowrange from about 0.1 ppm to about 10 ppm. Alternative ranges of umamiflavor enhancing amounts can be from about 0.01 ppm to about 30 ppm,from about 0.05 ppm to about 15 ppm, from about 0.1 ppm to about 5 ppm,or from about 0.1 ppm to about 3 ppm.

A “T1R1/T1R3 receptor modulating or activating amount” is an amount ofcompound that is sufficient to modulate or activate a T1R1/T1R3receptor. These amounts are preferably the same as the umami receptormodulating amounts.

An “umami receptor” is a taste receptor that can be modulated by asavory compound. Preferably an umami receptor is a G protein coupledreceptor, and more preferably the umami receptor is a T1R1/T1R3receptor.

Compounds of the inventions described herein modulate an umami receptorand preferably are agonists of the T1R1/T1R3 receptor. An agonist ofthis receptor has the effect of activating a G protein signalingcascade. In many cases, this agonist effect of the compound on thereceptor also produces a perceived savory flavor in a taste test. It isdesirable, therefore, that such inventive compounds serve as areplacement for MSG, which is not tolerated by some in, for example,comestible products.

In addition, this agonist effect also is responsible for the synergisticsavory taste effect, which occurs when a compound of the invention iscombined with another savory flavoring agent such as MSG. Thenucleotides, IMP or GMP, are conventionally added to MSG, to intensifythe savory flavor of MSG, so that relatively less MSG is needed toprovide the same savory flavor in comparison to MSG alone. Therefore, itis desirable that combining compounds of the inventions with anothersavory flavoring agent such as MSG into a comestible composition orformulation advantageously eliminates the need to add expensivenucleotides, such as IMP, as a flavor enhancer, while concomitantlyreducing or eliminating the amount of a savory compound such as MSGneeded to provide the same savory flavor in comparison to the savorycompound or MSG alone.

A “synergistic effect” relates to the enhanced savory flavor of acombination of savory compounds in comparison to the sum of the tasteeffects or flavor associated effects associated with each individualcompound. In the case of savory enhancer compounds, a synergistic effecton the effectiveness of MSG may be indicated for a compound of Formula(I) having an EC50 ratio (defined hereinbelow) of 2.0 or more, orpreferably 5.0 or more, or 10.0 or more, or 15.0 or more.

“Homogenization,” as the term is used herein, refers to any process foraltering particle or droplet size (e.g., reducing size and/or creatingsize uniformity) in a fluid under conditions of pressure, shear, and/orstress. The term “homogenization” is intended to include the many andvaried homogenization processes that involve the use of ultrasonic,pressure, and/or mechanical forces to homogenize a fluid. Examples ofsuch homogenization techniques include, but are not limited to,two-stage homogenization, high-pressure homogenization (also known asmicronization), very high pressure homogenization (VPH), rotator-statorhomogenization, blade homogenization, high shear mixers, sonication,high shear impellers, milling, and the like.

When the compounds described here include one or more chiral centers,the stereochemistry of such chiral centers can independently be in the Ror S configuration, or a mixture of the two. The chiral centers can befurther designated as R or S or R,S or d,D, l,L or d,l, D,L.Correspondingly, the amide compounds of the invention, if they can bepresent in optically active form, can actually be present in the form ofa racemic mixture of enantiomers, or in the form of either of theseparate enantiomers in substantially isolated and purified form, or asa mixture comprising any relative proportions of the enantiomers.

As used herein, “hydrocarbon residue” refers to a chemical sub-groupwithin a larger chemical compound which has only carbon and hydrogenatoms. The hydrocarbon residue may be aliphatic or aromatic,straight-chain, cyclic, branched, saturated or unsaturated. Thehydrocarbon residue, when so stated however, may contain or besubstituted with heteroatoms such as O, S or N, or the halogens(fluorine, chlorine, bromine, and iodine), or substituent groupscontaining heteroatoms (OH, NH₂, NO₂, SO₃H, and the like) over and abovethe carbon and hydrogen atoms of the substituent residue. Thus, whenspecifically noted as containing such heteroatoms, or designated as“substituted,” the hydrocarbon residue may also contain carbonyl groups,amino groups, hydroxyl groups and the like, or contain heteroatomsinserted into the “backbone” of the hydrocarbon residue.

As used herein, “inorganic residue” refers to a residue that does notcontain carbon, but contains at least some heteroatoms, including O, N,S, one or more halogens, or alkali metal or alkaline earth metal ions.Examples include, but are not limited to H, Na+, Ca++ and K+, halo,hydroxy, NO₂ or NH₂.

As used herein, the term “alkyl,” “alkenyl” and “alkynyl” includestraight- and branched-chain and cyclic monovalent substituents thatrespectively are saturated, unsaturated with at least one double bond,and unsaturated with at least one triple bond.

“Alkyl” refers to a hydrocarbon group that can be conceptually formedfrom an alkane by removing hydrogen from the structure of a hydrocarboncompound having straight or branched carbon chains, and replacing thehydrogen atom with another atom or substitutent group. In someembodiments of the invention, the alkyl groups are “C1 to C6 alkyl” suchas methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl,tert-butyl, amyl, tert-amyl, hexyl and the like. In some embodiments ofthe invention “C1 to C4 alkyl” groups (alternatively termed “loweralkyl” groups are methyl, ethyl, propyl, iso-butyl, sec-butyl t-butyl,and iso-propyl. Some of the preferred alkyl groups of the invention havethree or more carbon atoms preferably 3 to 16 carbon atoms, 4 to 14carbon atoms, or 6 to 12 carbon atoms.

Preferred alkenyl groups are “C2 to C7 alkenyl” such as vinyl, allyl,2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl,3-hexenyl, 4-hexenyl, 5-hexenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl,5-heptenyl, 6-heptenyl, as well as dienes and trienes of straight andbranched chains.

Preferred alkynyl groups are “C2 to C7 alkynyl” such as ethynyl,propynyl, 2-butynyl, 2-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl,4-hexynyl, 2-heptynyl, 3-heptynyl, 4-heptynyl, 5-heptynyl as well as di-and tri-ynes of straight and branched chains.

Hydrocarbon residues may be optionally substituted. Two of said optionalsubstituents on adjacent positions can be joined to form a fused,optionally substituted aromatic or nonaromatic, saturated or unsaturatedring which contains 3-8 members. Optional substituents are generallyhydrocarbon residues that may contain one or more heteroatoms or aninorganic residue such as H, Na⁺, Ca²⁺ or K⁺.

The terms “substituted alkyl,” “substituted alkenyl,” “substitutedalkynyl,” and “substituted alkylene” denote that the alkyl, alkenyl,alkynyl and alkylene groups are substituted by one or more, andpreferably one or two substituents, preferably halogen, hydroxy, C1 toC7 alkoxy, alkoxy-alkyl, oxo, C₃ to C₇ cycloalkyl, naphthyl, amino,(monosubstiruted)amino, (disubstituted)amino, guanidino, heterocycle,substituted heterocycle, imidazolyl, indolyl, pyrrolidinyl, C1 to C7acyl, C1 to C7 acyloxy, nitro, carboxy, carbamoyl, carboxamide, N-(Ci toC6 alkyl)carboxamide, N,N-di(C1 to C6 alkyl)carboxamide, cyano,methylsulfonylamino, thiol, C1 to C4 alkylthio or C1 to C4 alkylsulfonylgroups. The substituted alkyl groups may be substituted once or more,and preferably once or twice, with the same or with differentsubstituents. In many embodiments of the invention, a preferred group ofsubstituent groups include hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂,CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl, trifluoromethyl,methoxy, ethoxy, isopropoxy, and trifluoromethoxy groups. In manyembodiments of the invention that comprise the above lists ofsubstituent groups, an even more preferred group of substituent groupsinclude hydroxy, SEt, SCH₃, methyl, ethyl, isopropyl, methoxy, andethoxy groups.

Examples of the above substituted alkyl groups include the2-oxo-prop-1-yl, 3-oxo-but-1-yl, cyanomethyl, nitromethyl, chloromethyl,hydroxymethyl, tetrahydropyranyloxymethyl, trityloxymethyl,propionyloxymethyl, aminomethyl, carboxymethyl, allyloxycarbonylmethyl,allyloxycarbonylaminomethyl, methoxymethyl, ethoxymethyl,t-butoxymethyl, acetoxymethyl, chloromethyl, trifluoromethyl,6-hydroxyhexyl, 2,4-dichloro(n-butyl), 2-aminopropyl, 1-chloroethyl,2-chloroethyl, 1-bromoethyl, 2-chloroethyl, 1-fluoroethyl,2-fluoroethyl, 1-iodoethyl, 2-iodoethyl, 1-chloropropyl, 2-chloropropyl,3-chloropropyl, 1-bromopropyl, 2-bromopropyl, 3-bromopropyl,1-fluoropropyl, 2-fluoropropyl, 3-fluoropropyl, 2-aminoethyl,1-aminoethyl, N-benzoyl-2-aminoethyl, N-acetyl-2-aminoethyl,N-benzoyl-1-aminoethyl, N-acetyl-1-aminoethyl and the like.

Examples of the above substituted alkenyl groups include styrenyl,3-chloro-propen-1-yl, 3-chloro-buten-1-yl, 3-methoxy-propen-2-yl,3-phenyl-buten-2-yl, 1-cyano-buten-3-yl and the like. The geometricalisomerism is not critical, and all geometrical isomers for a givensubstituted alkenyl can be used.

Examples of the above substituted alkynyl groups includephenylacetylen-1-yl, 1-phenyl-2-propyn-1-yl and the like.

The term “oxo” denotes a carbon atom bonded to two additional carbonatoms substituted with an oxygen atom doubly bonded to the carbon atom,thereby forming a ketone moiety.

“Alkoxy” refers to an OR group, wherein R is an alkyl or substitutedalkyl. “Alkoxy-alkyl” refers to an alkyl group containing an alkoxy.

Preferred alkoxy groups are “C1 to C7 alkoxy” such as methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, t-butoxy and like groups. The term “C1to C7 substituted alkoxy” means the alkyl portion of the alkoxy can besubstituted in the same manner as in relation to C1 to C6 substitutedalkyl. Similarly, the term “C1 to C7 phenylalkoxy” as used herein means“C1 to C7 alkoxy” bonded to a phenyl radical.

“Acyloxy” refers to an OR group where R is an acyl group. Preferredacyloxy groups are “C1 to C7 acyloxy” such as formyloxy, acetoxy,propionyloxy, butyryloxy, pivaloyloxy, pentanoyloxy, hexanoyloxy,heptanoyloxy and the like.

As used herein, “acyl” encompasses the definitions of alkyl, alkenyl,alkynyl and the related hetero-forms which are coupled to an additionalresidue through a carbonyl group. Preferred acyl groups are “C1 to C7acyl” such as formyl, acetyl, propionyl, butyryl, pentanoyl, pivaloyl,hexanoyl, heptanoyl, benzoyl and the like. More preferred acyl groupsare acetyl and benzoyl.

Cycloalkyl residues are hydrocarbon groups within a molecule thatcomprise at least one ring having 3 to 8 carbon atoms linked into aring. Examples of such cyclalkyl residues include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl rings, andsaturated bicyclic or fused polycyclic cycloalkanes such as decalingroups, norborayl groups, and the like. Preferred cycloalkyl groupsinclude “C₃ to C₇ cycloalkyl” such as cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl or cycloheptyl rings. Similarly, the term “C₅ toC₇ cycloalkyl” includes the cyclopentyl, cyclohexyl or cycloheptylrings.

“Substituted cycloalkyl” indicates the above cycloalkyl rings aresubstituted preferably by one or two halogen, hydroxy, C1 to C4alkylthio, C1 to C4 alkylsulfoxide, C1 to C4 alkylsulfonyl, C1 to C4substituted alkylthio, C1 to C4 substituted alkylsulfoxide, C1 to C4substituted alkylsulfonyl, C1 to C6 alkyl, C1 to C7 alkoxy, C1 to C6substituted alkyl, C1 to C7 alkoxy-alkyl, oxo (monosubstituted)amino,(disubstituted)amino, trifluoromethyl, carboxy, phenyl, substitutedphenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino. In manyembodiments of substituted cycloalkyl groups, the substituted cycloalkylgroup will have 1, 2, 3, or 4 substituent groups independently selectedfrom hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃,methyl, ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, and trifluoromethoxy groups.

The term “cycloalkylene” means a cycloalkyl, as defined above, where thecycloalkyl radical is bonded at two positions connecting together twoseparate additional groups. Similarly, the term “substitutedcycloalkylene” means a cycloalkylene where the cycloalkyl radical isbonded at two positions connecting together two separate additionalgroups and further bearing at least one additional substituent.

The term “cycloalkenyl” indicates preferably a 1,2, or 3-cyclopentenylring, a 1,2,3 or 4-cyclohexenyl ring or a 1,2,3,4 or 5-cycloheptenylring, while the term “substituted cycloalkenyl” denotes the abovecycloalkenyl rings substituted with a substituent, preferably by a C1 toC6 alkyl, halogen, hydroxy, C1 to C7 alkoxy, alkoxy-alkyl,trifluoromethyl, carboxy, alkoxycarbonyl oxo, (monosubstituted)amino,(disubstituted)amino, phenyl, substituted phenyl, amino, or protectedamino.

The term “heterocycle” or “heterocyclic ring” denotes optionallysubstituted 3 to 8-membered rings having one or more carbon atomsconnected in a ring that also have 1 to 5 heteroatoms, such as oxygen,sulfur and/or nitrogen inserted into the ring. These 3 to 8-memberedrings may be saturated, unsaturated or partially unsaturated, but arepreferably saturated. An “amino-substituted heterocyclic ring” means anyone of the above-described heterocyclic rings is substituted with atleast one amino group. Preferred heterocyclic rings include furanyl,thiofuranyl, piperidyl, pyridyl, morpholino, aziridinyl, piperidinyl,piperazinyl, tetrahydrofurano, pyrrolo, and tetrahydrothiophen-yl.

The term “substituted heterocycle” or “substituted heterocyclic ring”means the above-described heterocyclic ring is substituted with, forexample, one or more, and preferably one or two, substituents which arethe same or different which substituents preferably can be halogen,hydroxy, thio, alkylthio, cyano, nitro, C1 to C6 alkyl, C1 to C7 alkoxy,C1 to C7 substituted alkoxy, alkoxy-alkyl, C1 to C7 acyl, C1 to C7acyloxy, carboxy, alkoxycarbonyl, carboxymethyl, hydroxymethyl,alkoxy-alkyl amino, monosubstituted)amino, (disubstituted)aminocarboxamide, N—(C1 to C6 alkyl)carboxamide, N,N-di(C1 to C6alkyl)carboxamide, trifluoromethyl, N—((C1 to C6 alkyl)sulfonyl)amino,N-(phenylsulfonyl)amino groups, or substituted with a fused ring, suchas benzo-ring. In many embodiments of substituted heterocyclic groups,the substituted cycloalkyl group will have 1, 2, 3, or 4 substituentgroups independently selected from hydroxy, fluoro, chloro, NH₂, NHCH₃,N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups.

An “aryl” groups refers to a monocyclic aromatic, linked bicyclicaromatic or fused bicyclic aromatic moiety comprising at least one sixmembered aromatic “benzene” ring, preferably comprising between 6 and 12ring carbon atoms, such as phenyl, biphenyl or naphthyl groups, whichmay be optionally substituted with various organic and/or inorganicsubstitutent groups, wherein the substituted aryl group and itssubstituents comprise between 6 and 18, or preferably 6 and 16 totalcarbon atoms. Preferred optional substituent groups include 1, 2, 3, or4 substituent groups independently selected from hydroxy, fluoro,chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy groups.

The term “heteroaryl” means a heterocyclic aryl derivative whichpreferably contains a five-membered or six-membered conjugated andaromatic ring system having from 1 to 4 heteroatoms, such as oxygen,sulfur and/or nitrogen, inserted into the unsaturated and conjugatedheterocyclic ring. Heteroaryl groups include monocyclic heteroaromatic,linked bicyclic heteroaromatic or fused bicyclic heteroaromaticmoieties. Examples of heteroaryls include pyridinyl, pyrimidinyl, andpyrazinyl, pyridazinyl, pyrrolyl, furanyl, thiofuranyl, oxazoloyl,isoxazolyl, phthalimido, thiazolyl, quinolinyl, isoquinolinyl, indolyl,or a furan or thiofuran directly bonded to a phenyl, pyridyl, orpyrrolyl ring and like unsaturated and conjugated heteroaromatic rings.Any monocyclic, linked bicyclic, or fused bicyclic heteroaryl ringsystem which has the characteristics of aromaticity in terms of electrondistribution throughout the ring system is included in this definition.Typically, the heteroaromatic ring systems contain 3-12 ring carbonatoms and 1 to 5 ring heteroatoms independently selected from oxygen,nitrogen, and sulfur atoms.

The term “substituted heteroaryl” means the above-described heteroarylis substituted with, for example, one or more, and preferably one ortwo, substituents which are the same or different which substituentspreferably can be halogen, hydroxy, protected hydroxy, thio, alkylthio,cyano, nitro, C1 to C6 alkyl, C1 to C7 substituted alkyl, C1 to C7alkoxy, C1 to C7 substituted alkoxy, alkoxy-alkyl, C1 to C7 acyl, C1 toC7 substituted acyl, C1 to C7 acyloxy, carboxy, alkoxycarbonyl,carboxymethyl, hydroxymethyl, amino, (monosubstituted)amino,(disubstituted)amino, carboxamide, N—(C1 to C6 alkyl)carboxamide,N,N-di(C1 to C6 alkyl)carboxamide, trifluoromethyl, N—((C1 to C6alkyl)sulfonyl)ammo or N-(phenylsulfonyl)amino groups. In manyembodiments of substituted heteroaryl groups, the substituted cycloalkylgroup will have 1, 2, 3, or 4 substituent groups independently selectedfrom hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CBb)₂, CO₂CH₃, SEt, SCH₃,methyl, ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, and trifluoromethoxy groups.

Examples of the term “substituted arylalkyl” include groups such as2-phenyl-1-chloroethyl, 2-(4-methoxyphenyl)ethyl, 4-(2,6-dihydroxyphenyl)-n-hexyl, 2-(5-cyano-3-methoxyphenyl)-n-pentyl,3-(2,6-dimethylphenyl)propyl, 4-chloro-3-aminobenzyl,6-(4-methoxyphenyl)-3-carboxy-n-hexyl,5-(4-aminomethylphenyl)-3-(aminomethyl)-n-pentyl,5-phenyl-3-oxo-n-pent-1-yl and the like.

The term “arylalkylene” specifies an arylalkyl, as defined above, wherethe arylalkyl radical is bonded at two positions connecting together twoseparate additional groups. The definition includes groups of theformula: -phenyl-alkyl- and alkyl-phenyl-alkyl-. Substitutions on thephenyl ring can be 1,2, 1, 3 or 1,4. The term “substituted arylalkylene”is an arylalkylene as defined above that is further substitutedpreferably by halogen, hydroxy, protected hydroxy, C1 to C4 alkylthio,C1 to C4 alkylsulfoxide, C1 to C4 alkylsulfonyl, C1 to C4 substitutedalkylthio, C1 to C4 substituted alkylsulfoxide, C1 to C4 substitutedalkylsulfonyl, C1 to C6 alkyl, C1 to C7 alkoxy, C1 to C6 substitutedalkyl, C1 to C7 alkoxy-alkyl, oxo, (tnonosubstituted)amino,(disubstituted)amino, trifluoromethyl, carboxy, alkoxycarbonyl, phenyl,substituted phenyl, phenylthio, phenylsulfoxide, phenylsulfonyl, amino,or protected amino group on the phenyl ring or on the alkyl group.

The term “substituted phenyl” specifies a phenyl group substituted withone or more, and preferably one or two, moieties preferably chosen fromthe groups consisting of halogen, hydroxy, protected hydroxy, thio,alkylthio, cyano, nitro, C1 to C6 alkyl, C1 to C6 substituted alkyl, C1to C7 alkoxy, C1 to C7 substituted alkoxy, alkoxy-alkyl, C1 to C7 acyl,C1 to C7 substituted acyl, C1 to C7 acyloxy, carboxy, alkoxycarbonyl,carboxymethyl, hydroxymethyl, amino, (monosubstituted)amino,(disubstituted)amino, carboxamide, N—(C1 to C6 alkyl)carboxamide,N,N-di(C1 to C6 alkyl)carboxamide, trifluoromethyl, N—((C1 to C6alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl, wherein thephenyl is substituted or unsubstituted, such that, for example, abiphenyl results. In many embodiments of substituted phenyl groups, thesubstituted cycloalkyl group will have 1, 2, 3, or 4 substituent groupsindependently selected from hydroxy, fluoro, chloro, NH₂, NHCH₃,N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo atoms. There can be one or more halogen, which are the same ordifferent. Preferred halogens are chloro and fluoro. Although many ofthe compounds of the invention having halogen atoms as substituents arevery effective in binding to the relevant taste receptors, suchhalogenated organic compounds can often have undesirable toxicologicalproperties when administered to an animal in vivo. Therefore, in manyembodiments of the compounds of Formula (I), if a halogen atom(including a fluoro or chloro atom) is listed as a possible substitutentatom, an alternative preferred group of substitutents would NOT includethe halogen, fluorine, or chlorine groups.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent preferably chosen from the group consisting of phenyl,substituted phenyl, C1 to C6 alkyl, C1 to C6 substituted alkyl, C1 to C7acyl, C1 to C7 substituted acyl, C2 to C7 alkenyl, C2 to C7 substitutedalkenyl, C2 to C7 alkynyl, C2 to C7 substituted alkynyl, C7 to C12phenylalkyl, C7 to C12 substituted phenylalkyl and heterocyclic ring.The (monosubstituted)amino can additionally have an amino-protectinggroup as encompassed by the term “protected (monosubstituted)amino.”

The term “(disubstituted)amino” refers to an amino group substitutedpreferably with two substituents chosen from the group consisting ofphenyl, substituted phenyl, C1 to C6 alkyl, C1 to C6 substituted alkyl,C1 to C7 acyl, C2 to C7 alkenyl, C2 to C7 alkynyl, C7 to C12phenylalkyl, and C7 to C12 substituted phenylalkyl. The two substituentscan be the same or different.

The term “substituted alkylene” means an alkyl group where the alkylradical is bonded at two positions connecting together two separateadditional groups and further bearing an additional substituent.Examples of “substituted alkylene” includes aminomethylene,1-(amino)-1,2-ethyl, 2-(amino)-1,2-ethyl, 1-(acetamido)-1,2-ethyl,2-(acetamido)-1,2-ethyl, 2-hydroxy-1,1-ethyl, 1-(amino)-1,3-pκ>pyl.

One or more of the compounds of the invention, may be present as a salt.The term “salt” encompasses those salts that form with the carboxylateanions and amine nitrogens and include salts formed with the organic andinorganic anions and cations discussed below. Furthermore, the termincludes salts that form by standard acid-base reactions with basicgroups (such as amino groups) and organic or inorganic acids. Such acidsinclude hydrochloric, hydrofluoric, trifluoroacetic, sulfuric,phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic,cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic,tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic,sorbic, picric, benzoic, cinnamic, and like acids.

The term “organic or inorganic cation” refers to counter-ions for thecarboxylate anion of a carboxylate salt. The counter-ions are chosenfrom the alkali and alkaline earth metals, (such as lithium, sodium,potassium, barium, aluminum and calcium); ammonium and mono-, di- andtri-alkyl amines such as trimethylamine, cyclohexylamine; and theorganic cations, such as dibenzylammonium, benzylammonium,2-hydroxyethylammonium, bis(2-hydroxyethyl) ammonium,phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations.See, for example, “Pharmaceutical Salts,” Berge, et al., J. Pharm. Sci.(1977) 66:1-19, which is incorporated herein by reference. Other cationsencompassed by the above term include the protonated form of procaine,quinine and N-methylglucosamine, and the protonated forms of basic aminoacids such as glycine, ornithine, histidine, phenylglycine, lysine andarginine. Furthermore, any zwitterionic form of the instant compoundsformed by a carboxylic acid and an amino group is referred to by thisterm. For example, a cation for a carboxylate anion will exist when R2or R3 is substituted with a (quaternary ammonium)methyl group. Apreferred cation for the carboxylate anion is the sodium cation.

The compounds of the invention can also exist as solvates and hydrates.Thus, these compounds may crystallize with, for example, waters ofhydration, or one, a number of, or any fraction thereof of molecules ofthe mother liquor solvent. The solvates and hydrates of such compoundsare included within the scope of this invention.

The term “amino acid” includes any one of the twenty naturally-occurringamino acids or the D-form of any one of the naturally-occurring aminoacids. In addition, the term “amino acid” also includes othernon-naturally occurring amino acids besides the D-amino acids, which arefunctional equivalents of the naturally-occurring amino acids. Suchnon-naturally-occurring amino acids include, for example, norleucine(“NIe”), norvaline (“Nva”), L- or D-naphthalanine, ornithine (“Orn”),homoarginine (homoArg) and others well known in the peptide art, such asthose described in M. Bodanzsky, “Principles of Peptide Synthesis,” 1stand 2nd revised ed., Springer-Verlag, New York, N.Y., 1984 and 1993, andStewart and Young, “Solid Phase Peptide Synthesis,” 2nd ed., PierceChemical Co., Rockford, Ill., 1984, both of which are incorporatedherein by reference. Amino acids and amino acid analogs can be purchasedcommercially (Sigma Chemical Co.; Advanced Chemtech) or synthesizedusing methods known in the art.

“Amino acid side chain” refers to any side chain from theabove-described “amino acids.”

“Substituted” herein refers to a substituted moiety, such as ahydrocarbon, e.g., substituted alkyl or benzyl wherein at least oneelement or radical, e.g., hydrogen, is replaced by another, e.g., ahydrogen is replaced by a halogen as in chlorobenzyl.

A residue of a chemical species, as used in the specification andconcluding claims, refers to a structural fragment, or a moiety that isthe resulting product of the chemical species in a particular reactionscheme or subsequent formulation or chemical product, regardless ofwhether the structural fragment or moiety is actually obtained from thechemical species. Thus, an ethylene glycol residue in a polyester refersto one or more —OCH₂CH₂O— repeat units in the polyester, regardless ofwhether ethylene glycol is used to prepare the polyester. Similarly, a2,4-thiazolidinedione residue in a chemical compound refers to one ormore-2,4-thiazolidinedione moieties of the compound, regardless ofwhether the residue was obtained by reacting 2,4-thiazolidinedione toobtain the compound.

The term “organic residue” defines a carbon containing residue, i.e. aresidue comprising at least one carbon atom, and includes but is notlimited to the carbon-containing groups, residues, or radicals definedhereinabove. Organic residues can contain various heteroatoms, or bebonded to another molecule through a heteroatom, including oxygen,nitrogen, sulfur, phosphorus, or the like. Examples of organic residuesinclude but are not limited alkyl or substituted alkyls, alkoxy orsubstituted alkoxy, mono or di-substituted amino, amide groups, etc.Organic resides can preferably comprise 1 to 18 carbon atoms, 1 to 15,carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, or 1 to 4carbon atoms.

By the term “effective amount” of a compound as provided herein is meanta sufficient amount of the compound to provide the desired regulation ofa desired function, such as activation of a taste receptor, or to causeperception of taste. As will be pointed out below, the exact amountrequired will vary from subject to subject, depending on the species,age, general condition of the subject, specific identity and formulationof the drug, etc. Thus, it is not possible to specify an exact“effective amount.” However, an appropriate effective amount can bedetermined by one of ordinary skill in the art using only routineexperimentation.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an aromatic compound” includes mixtures of aromaticcompounds.

Often, ranges are expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally substituted lower alkyl”means that the lower alkyl group may or may not be substituted and thatthe description includes both unsubstituted lower alkyl and lower alkylswhere there is substitution.

The Savory Amide Compounds to be Formulated

Many of the high potency savory compounds employed in formulating thecomestible compositions described herein include the “amide” compoundsthat were recently disclosed in U.S. Patent Publication No. US2005/0084506 A1 and U.S. Patent Publication No. US 2006/0045953 A1, bothof which applications are hereby incorporated by reference herein intheir entirety, for all purposes, but particularly for their descriptionof “amide” compounds that have an extremely high potency as Umamifiavorant compounds, methods and examples of their synthesis, and dataon their biological effectiveness as Umami flavorants or tastants. Manyof the savory amide compounds used in the comestible compositions andformulations described herein are organic (carbon containing) compoundsthat all have at least one “amide” group therein, have the followinggeneral structure, which will be hereinafter referred to as the amidecompounds having Formula (I) shown below:

The amide compounds of Formula (I) do not include any amide compoundsthat are known to naturally occur in biological systems or foods, suchas peptides, proteins, nucleic acids, glycopeptides or glycoproteins, orthe like. The amide compounds of Formula (I) of the invention areman-made and artificial synthetic amide compounds.

For the various embodiments of the compounds of Formula (I), the R¹, R²and R³ groups can be and are independently further defined in many ways,for example as was described in detail in U.S. Patent Publication No. US2005/0084506 A1 and U.S. Patent Publication No. US 2006/0045953 A1,hereby incorporated herein by reference for their disclosures relatingto the structures and biological activity of the amide compoundsdescribed herein, and methods for their synthesis and purification. Itis hereby specifically contemplated that any of subgenuses and/orspecies of compounds of Formula (I) described in U.S. Patent PublicationNo. US 2005/0084506 A1 and U.S. Patent Publication No. US 2006/0045953A1 can be employed in the compositions, processes and/or methodsdescribed herein below, to form a savory or sweet flavor modifiedcomestible or medicinal product, or a precursor thereof.

In many aspects of the compounds of Formula (T), R¹ comprises an organicor hydrocarbon-based residue having at least three carbon atoms andoptionally one to 20, 15, 10, 8, 7, 6, or 5 heteroatoms independentlyselected from oxygen, nitrogen, sulfur, halogens, or phosphorus.

In many aspects of the compounds of Formula (I), one of R² and R³ isoptionally H, and one or both of R² and R³ comprises an organic orhydrocarbon-based residue having at least three carbon atoms andoptionally one to ten heteroatoms independently selected from oxygen,nitrogen, sulfur, halogens, or phosphorus.

In many aspects of the compounds of Formula (I), the molecular weight ofthe compounds of Formula (I) should be less than about 800 grams permole, or in further related embodiments less than or equal to about 700grams per mole, 600 grams per mole, 500 grams per ole, 450 grams permole, 400 grams per mole, 350 grams per mole, or 300 grams per mole.Similarly, the compounds of Formula (I) can have preferred ranges ofmolecular weight, such as for example from about 175 to about 500 gramsper mole, from about 200 to about 450 grams per mole, from about 225 toabout 400 grams per mole, from about 250 to about 350 grams per mole.

For example, in some aspects, R¹, R², and R³ can be independentlyselected from the group consisting of an arylalkenyl, heteroarylalkenyl,arylalkyl, heteroarylalkyl, alkyl, alkoxy alkyl, alkenyl, cycloalkyl,cycloalkenyl, aryl, heteroaryl, —R⁴OH, —R⁴OR⁵, —R⁴CN, —R⁴CO₂H, —R⁴CO₂R⁵,—R⁴COR⁵, —R⁴SR⁵, and —R⁴SO₂R⁵, and optionally substituted derivativethereof comprising 1, 2, 3, or 4 carbonyl, amino groups, hydroxyl, orhalogen groups, and wherein R⁴ and R⁵ are C₁-C₆ hydrocarbon residues.

In further related embodiments of the amide compounds of Formula (I),R¹, R² and R³ can be independently selected from the group consisting ofan arylalkenyl, heteroarylalkenyl, arylalkyl, heteroarylalkyl, alkyl,alkoxy-alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycle, aryl andheteroaryl groups, and optionally substituted derivatives thereofcomprising 1, 2, 3 or 4 carbonyl, amino groups, hydroxyl, chlorine, orfluorine groups. In both of the embodiments just mentioned, analternative and preferred set of optional substituent groups would besubstituents independently selected from hydroxy, fluoro, chloro, NH₂,NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxysubstituent groups.

In many aspects of the compounds of Formula (I), one of R² and R³ ishydrogen and the other R² or R³ group is an organic residue or group.For example, in many embodiments of the compounds of Formula (I), atleast one or R² and R³ is a branched or cyclic organic residue having acarbon atom directly bonded to both (a) the amide nitrogen atom and (b)two additional carbon atoms from other organic residues, which arebranched or cyclic organic residues comprising additional hydrogen atomsand up to 10 optional additional carbon atoms, and optionally from zeroto five heteroatoms independently selected from oxygen, nitrogen,sulfur, fluorine, and chlorine. Such branched R² and R³ groups includeorganic radicals having the formula:

wherein n_(a) and n_(b) are independently selected from 1, 2, and 3, andeach R^(2a) or R^(2b) substituent residue is independently selected fromhydrogen, a halogen, a hydroxy, or a carbon-containing residueoptionally having from zero to five heteroatoms independently selectedfrom oxygen, nitrogen, sulfur, and a halogen. In some such embodiments,the R^(2a) or R^(2b) are independent substituent groups, but in otherembodiments one or more of the R^(2a) or R^(2b) radicals can be bondedtogether to form ring structures.

In some aspects of the compounds of Formula (I), at least one of the R²and R³ is a branched alkyl radical having 5 to 12 carbon atoms, or atleast one of R² and R³ is a cycloalkyl or cycloalkenyl ring comprising 5to 12 ring carbon atoms. In such embodiments of R² and R³ the branchedalkyl radical or the cycloalkyl or cycloalkenyl ring can be optionallysubstituted with 1, 2, 3, or 4 substituent groups independently selectedfrom hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH, SR⁹, halogen, CN, NO₂,CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰, NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰,NR⁹SO₂R¹⁰, alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, andheterocycle; and R⁹ and R¹⁰ are independently selected from H, alkyl,cycloalkyl, and alkenyl. In some related aspects, the substituents forthe compounds can be selected from hydroxy, fluoro, chloro, NH₂, NHCH₃,N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxy.

In many aspects of the compounds of Formula (I), at least one of R² orR³ is a C₃-C₁₀ or C₃-C₁₅ branched alkyl. These branched alkyls have beenfound to be highly effective R² groups for savory amide compounds. Infurther aspects the C₃-C₁₀ branched alkyl may optionally substitutedwith one or two substituents independently selected from a. hydroxy,fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy group.

In some aspects of the compounds of Formula (I), at least one of R² orR³ is a cycloalkyl, cycloalkenyl, or saturated heterocyclic ring having3 to 10 ring carbon atoms, optionally substituted with 1, 2, or 3substituents independently selected from the group consisting OfNH₂,NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, C₁-Q alkyl, C₁-C₄ haloalkyl, C₁-C₄alkoxy, C₁-C₄ haloalkoxy, hydroxy, and halogen. In some furtherembodiments, at least one of R² or R³ is a cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl ring, or piperidyl ring optionally substitutedwith 1, 2, or 3 substituents independently selected from the groupconsisting of hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt,SCH₃, methyl, ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, and trifluoromethoxy.

In some aspects, at least one of R² or R³ is a cyclohexyl, optionallysubstituted with 1, 2, or 3 methyl groups. Examples of such methylsubstituted cyclohexyl rings have the formula

In some aspects of the compounds of Formula (I), at least one of R² orR³ is a 1-(1,2,3,4) tetrahydronapthalene ring or an2,3-dihydro-1H-indene ring having the formula:

wherein m is 0, 1, 2, or 3, and each R² can be bonded to either thearomatic or non-aromatic ring and is independently selected fromhydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy. It is to be understood that optical and/ordiastereomeric isomerism can occur on the cyclohexyl or cyclopentylrings of these substituent, and differing optical and/or diastereomerscan often have at least somewhat differing biological activities.Aromatic or Heteroaromatic Compounds

In many aspects of the amide compounds of Formula (I) having savoryreceptor agonist activity, the invention relates to a subgenus ofaromatic amide compounds having the following formula (II):

wherein A comprises a 5 or 6 membered aryl or heteroaryl ring; m is 0,1, 2, 3 or 4; each R¹ is independently selected from alkyl, alkoxy,alkoxy-alkyl. hydroxyalkyl, OH, CN, CO₂H₅CO₂R⁶, CHO, COR⁶, SR⁶, halogen,alkenyl, cycloalkyl, cycloalkenyl, heterocycle, aryl, and heteroaryl;and R⁶ is C₁-C₆ alkyl, and R² can be any of the embodiments contemplatedherein above, or the like.

In many aspects, the A group of Formula (II) comprises an aryl ring,i.e. it contains at least one six-membered phenyl (benzene) ring. Thearyls include at least benzene and napthalene rings, which may not, butin many embodiments are further substituted with at least 1, 2, or 3 R¹substituent groups independently selected from the group consisting ofhydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, COOCH₃, SCH₃, SEt, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy groups.

In some preferred embodiments, one or two of the R¹ substituent groupsare bonded together to form a saturated alkylenedioxy ring on a phenylring, as exemplified by the following preferred structures (IIa) and(IIb);

wherein R_(1a), R_(1a′), and R_(1b) are independently hydrogen or alower alkyl, or alternatively R_(1a) and R_(1b) are independentlyhydrogen or methyl, or alternatively both R_(1a) and R_(1b) arehydrogen.

Additional examples of fused bicyclic heteroaryls as A groups aretypified by the following benzoxazole compounds (Formula IIe) and(Formula (IIf):

wherein R_(1a) or R_(1b) is independently hydrogen or a lower alkyl.

In many embodiments of the amide compounds of Formula (H), A is amonocyclic heteroaryl ring. The monocyclic heteroaryls that can be usedas an A group in Formula (II) are typified by the following structures:

wherein m is 0, 1, 2, or 3, and each R¹ is independently selected from,hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy.

In many embodiments of the compounds of the various subgenuses ofFormula (II) described immediately above, at least one of R¹ or R² canbe a C₃-C₁₅ branched alkyl; an α-substituted carboxylic acid or anα-substituted carboxylic acid lower alkyl ester; a 5 or 6 membered arylor heteroaryl ring, optionally substituted with 1, 2, 3 or 4 substituentgroups selected from the group consisting of hydroxy, fluoro, chloro,NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups; a cyclohexyl, optionally substituted with 1, 2, or 3 methylgroups; or a 1-(1,2,3,4) tetrahydronapthalene ring or an2,3-dihydro-1H-indene ring having the formula:

wherein m is 0, 1, 2, or 3, and each R^(2′) can be bonded to either thearomatic or non-aromatic ring and is independently selected fromhydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy; as were described hereinabove with respect to thegeneral amide compounds of Formula (I).

In many aspects of the inventions described herein, amide compoundswithin the subgenus of aromatic amide compounds described below can beemployed to formulate the comestible concentrate and/or final comestiblecompositions;

wherein

i) A is a 5 or 6 membered aryl or heteroaryl ring,

ii) m is 0, 1, 2, 3 or 4,

iii) each R^(1′) is independently selected from the group consisting ofhydroxyl, NH₂, SH, halogen, and a C₁-C₄ organic radical, and

iv) R² has 3 to 15 carbon atoms and is a branched alkyl, a cycloalkylring, or a heterocyclic ring, optionally substituted with 1, 2, 3, or 4substituent groups independently selected from the group consisting ofhydroxyl, NH2, SH, halogen, and a C₁-C₄ organic radical, or one or morecomestibly acceptable salts thereof,

The subgenuses of aromatic or heteroaromatic amide compounds of Formula(IT) described immediately above contain many excellent agonists ofT1R1/T1R3 savory (“umami”) taste receptors, that are effective to bindto the receptors at very low concentrations of the amide compound on theorder of micromolar concentrations or less, and can induce a noticeablesensation of a savory umami flavor in humans, and/or can serve asenhancers of the savory umami flavor of MSG.

Accordingly, many of the aromatic or heteroaromatic amide compounds ofFormula (II) can be utilized as savory flavoring agents or savory flavorenhancers when contacted with a wide variety of comestible productsand/or compositions, or their precursors, as is described elsewhereherein.

Oxalamide Compounds

In another subgenus of the amide compounds of Formula (I)₅ the amidecompound is an oxalamide compound having Formula (V):

wherein R¹⁰ and R³⁰ are each independently selected from a hydrocarbonresidue that may contain one or more heteroatoms, or preferably, R¹⁰ andR³⁰ are independently selected from the group consisting of arylalkyl,heteroarylalkyl, heterocycle-alkyl, or optionally substituted groupsthereof, and

R²⁰ and R⁴⁰ are each independently H or a hydrocarbon residue that maycontain one or more heteroatoms; preferably R²⁰ and R⁴⁰ are H or C₁-C₃alkyl, or optionally substituted groups thereof. More preferably R²⁰ andR⁴⁰ are H. Moreover, there can be 0, 1, 2, 3, or 4 optional substituentgroups for R¹⁰ and R³⁰ independently selected from hydroxy, fluoro,chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SCH₃, SEt, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy groups.

In preferred embodiments of the oxalamide compounds of Formula (V), R¹⁰and R³⁰ are independently selected hydrocarbon residues having at leastthree carbon atoms and optionally one to ten heteroatoms independentlyselected from oxygen, nitrogen, sulfur, halogens, or phosphorus, andwherein R²⁰ and R⁴⁰ are independently selected from hydrogen and ahydrocarbon residue having at least three carbon atoms and optionallyone to ten heteroatoms independently selected from oxygen, nitrogen,sulfur, halogens, or phosphorus.

In many preferred embodiments of the oxalamide compounds of Formula (V),R²⁰ and R⁴⁰ are hydrogen. Ih such embodiments, R¹⁰ and R³⁰ can beindependently selected from the group consisting of arylalkyls,heteroarylalkyls, cycloalkyl-alkyls, and heterocycle-alkyls comprisingfive to 15 carbon atoms, wherein each of R¹⁰ and R³⁰ can optionallycomprise one to one to four substituents independently selected fromhydrogen, hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt,SCH₃, methyl, ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy,isopropoxy, and trifluoromethoxy groups.

In many embodiments of the oxalamide compounds of Formula (V), theoxalamide compound has the Formula (Va):

wherein A and B are independently an aryl, heteroaryl, cycloalkyl, or aheterocycle comprising 5 to 12 ring atoms; m and n are independently 0,1, 2, 3 or 4-8; R²⁰ and R⁴⁰ are hydrogen, R⁵⁰ is hydrogen or an alkyl orsubstituted alkyl residue comprising one to four carbon atoms; R⁶⁰ isabsent or a C₁-C₅ alkylene or a C₁-C₅ substituted alkylene; R⁷⁰ and R⁸⁰are independently selected from the group consisting of hydrogen, alkyl,alkoxyl, alkoxy-alkyl, OH, SR⁹, halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰,NR⁹R¹⁰, NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle; R⁹ and R¹⁰are independently selected from H, Ci-C₆ alkyl, C₃-Q cycloalkyl, andCi-Cβ alkenyl.

In preferred embodiments of the oxalamide compounds of Formula (Va), R⁶⁰is a —CH₂CH₂. group, A and B are independently selected from phenyl,pyridyl, furanyl, thiofuranyl and pyrrolyl rings and R⁷⁰ and R⁸⁰ areindependently selected from hydroxy, fluoro, chloro, NH₂, NHCH₃,N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups.

In some embodiments of the oxalamide compounds of Formula (Va), A and Bare independently a phenyl, pyridyl, furanyl, benzofuranyl, pyrrole,benzothiophene, piperidyl, cyclopentyl, cyclohexyl, or cycloheptyl ring;m and n are independently 0, 1, 2, or 3; R²⁰ and R⁴⁰ are hydrogen; R⁵⁰is hydrogen or methyl; R⁶⁰ is a C₁-C₅ or preferably C₂ alkylene; R⁷⁰ andR⁸⁰ are independently selected from hydrogen, hydroxy, fluoro, chloro,NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups.

In many embodiments of the oxalamide compounds of Formula (V), theoxalamide compound has the Formula (Vb):

wherein A is a phenyl, pyridyl, furanyl, pyrrole, piperidyl,cyclopentyl, cyclohexyl, or cycloheptyl ring; m and n are independently0, 1, 2, or 3; R⁵⁰ is hydrogen or methyl; P is 1 or 2; and R⁷⁰ and R⁸⁰are independently selected from the group consisting of hydrogen,hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, COOCH₃, SCH₃, SEt, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy, or two of R⁷⁰ together form a methylenedioxy ring.In some embodiments of the oxalamide compounds of Formula (Vb), thepyridyl-R⁸⁰ radical has the structure:

In certain preferred embodiments of the amide compounds of Formula (V),the oxalamide compound has the Formula (Vc):

wherein Ar¹ is a substituted aryl or heteroaryl ring comprising five to12 carbon atoms; R⁵⁰ is hydrogen or methyl; n is 0, 1, 2, or 3; each R⁸⁰is independently selected from the group consisting of hydroxy, fluoro,chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy groups. In some embodiments of the oxalamide compoundsof Formula (Vc), Ar¹ is a 2-, 3-, or 4-mono-substituted phenyl, 2,4-,2,3-, 2,5,2,6,3,5-, or 3,6-disubstituted phenyl, 3-alkyl-4-substitutedphenyl, a tri-substituted phenyl wherein the substituent groups areindependently selected from the group consisting of hydrogen, hydroxy,fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy, or two adjacent substituents together form amethylenedioxy ring on the phenyl ring. In some embodiments of theoxalamide compounds of Formula (Vc), Ar¹ is a substituted heteroarylring comprising 5 to 12 carbon atoms and wherein the substituent groupsare independently selected from the group consisting of hydrogen,hydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy.

In certain preferred embodiments of the amide compounds of Formula (V),the oxalamide compound has the Formula (Vd):

wherein A is a substituted aryl or heteroaryl ring comprising five to 12carbon atoms; R⁵⁰ is hydrogen or methyl; n is 0, 1, 2, or 3; each R⁸⁰ isindependently selected from the group consisting of hydrogen, hydroxy,fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, COOCH₃, SCH₃, SEt, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy. Preferably, A is a phenyl, pyridyl, furanyl. pyrrole,piperidyl, cyclopentyl, cyclohexyl, or cycloheptyl ring optionallysubstituted with 1, 3, or 3 substituent groups independently selectedfrom the group consisting of hydrogen, hydroxy, fluoro, chloro, NH₂,NHCH₃, N(CH₃)₂, COOCH₃, SCH₃, SEt, methyl, ethyl, isopropyl, vinyl,trifluoromethyl, methoxy, ethoxy, isopropoxy, and trifluoromethoxygroups.

In some aspects of the amide compounds of Formula (V), the oxalamidecompound has the Formula (Ve):

wherein m and n are independently 0, 1, 2, or 3; R⁷⁰ and R⁸⁰ areindependently selected from the group consisting of hydrogen, alkyl,alkoxyl, alkoxy-alkyl, OH, SR⁹, halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰,NR⁹R¹⁰, NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle; and R⁹ andR¹⁰ are independently selected from H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,and C₁-C₆ alkenyl groups, hi related aspects, R⁷⁰ and R⁸⁰ areindependently selected from the group consisting of hydrogen, hydroxy,fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, COOCH₃, SCH₃, SEt, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy groups. Preferably, the pyridyl-R⁸⁰ radical of theoxalamide compound of Formula (Ve) has the structure:

Lastly, in some aspects related to processes for making savory flavorantconcentrate compositions described hereinbelow, another subgenus ofoxamamide compounds that includes numerous high potency agonists ofT1R1/T1R3 savory (“umami”) taste receptors include compounds having thefollowing structures:

wherein

i) A and B are independently an aryl, heteroaryl, cycloalkyl, orheterocycle comprising 5 to 12 ring atoms,

ii) m and n are independently 0, 1, 2, 3 or 4-8,

iii) R⁷⁰ and R₈₀ are independently selected from the group consisting ofhydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH, SR⁹, halogen, CN, NO₂,CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰, NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰,NR⁹SO₂R¹⁰, alkenyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, andheterocycle, and R⁹ and R¹⁰ are independently selected from H, C₁-C₆alkyl, C₃-C₆ cycloalkyl, and C₁-C₆ alkenyl, or two of R⁷⁰ together forma methylenedioxy ring, or one or more comestibly acceptable saltsthereof;

As can be noted by inspection of the Examples attached hereinbelow, theoxalamide compounds described above are excellent agonists of T1R1/T1R3savory (“umami”) taste receptors at very low concentrations on the orderof micromolar concentrations or less, and can induce a noticeablesensation of a savory umami flavor in humans, and/or can serve asenhancers of the savory umami flavor of MSG. Accordingly, oxalamidecompounds can be utilized as savory flavoring agents or savory flavorenhancers when contacted with a wide variety of comestible productsand/or compositions, or their precursors, as is described elsewhereherein.

Comestibly or Pharmaceutically Acceptable Compounds

Many of the amide compounds of Formula (I) or its various enumeratedsubgenuses comprise acidic or basic groups, so that depending on theacidic or basic character (“pH”) of the comestible or medicinalcompositions in which they are formulated, they may be present as salts,which are preferably comestibly acceptable (i.e. designated as generallyrecognized as safe, or GRAS) or pharmaceutically acceptable salts (manyof which have been recognized by the Federal Food and DrugAdministration).

The amide compounds of Formula (I) having acidic groups, such ascarboxylic acids, will tend (at near neutral physiological pH) to bepresent in solution in the form of anionic carboxylates, and thereforewill in preferred embodiments have an associate comestibly and/orpharmaceutically acceptable cation, many of which are known to those ofordinary skill in the art. Such comestibly and/or pharmaceuticallyacceptable cations include alkali metal cations (lithium, sodium, andpotassium cations), alkaline earth metal cations (magnesium, calcium,and the like), or ammonium (NH₄)⁺ or organically substituted ammoniumcations such as (R—NH₃)⁴⁺ cations.

The amide compounds of Formula (I) having basic substituent groups, suchas amino or nitrogen containing heterocyclic groups, will tend (at nearneutral physiological pH, or at the acidic pH common in many foods) tobe present in solution in the form of cationic ammonium groups, andtherefore will in preferred embodiments have an associate comestiblyand/or pharmaceutically acceptable anion, many of which are known tothose of ordinary skill in the art. Such comestibly and/orpharmaceutically acceptable anionic groups include the anionic form of avariety of carboxylic acids (acetates, citrates, tartrates, anionicsalts of fatty acids, etc.), halides (especially fluorides orchlorides), nitrates, and the like.

The amide compounds of Formula (I) and its various subgenuses shouldpreferably be comestibly acceptable, i.e. deemed suitable forconsumption in food or drink, and should also be pharmaceuticallyacceptable. The typical method of demonstrating that a fiavorantcompound is comestibly acceptable is to have the compound tested and/orevaluated by an Expert Panel of the Flavor and Extract ManufacturersAssociation and declared as to be “Generally Recognized As Safe”(“GRAS”). The FEMA/GRAS evaluation process for fiavorant compounds iscomplex but well known to those of ordinary skill in the food productpreparation arts, as is discussed by Smith et al. in an article entitled“GRAS Flavoring Substances 21,” Food Technology, 57(5), pgs 46-59, May2003, the entire contents of which are hereby incorporated herein byreference.

When being evaluated in the FEMA/GRAS process, a new flavorant compoundis typically tested for any adverse toxic effects on laboratory ratswhen fed to such rats for at least about 90 days at a concentration100-fold, or 1000-fold, or even higher concentrations than the proposedmaximum allowable concentration of the compound in a particular categoryof food products being considered for approval. For example, suchtesting of the amide compounds of the invention might involve combiningthe amide compound with rat chow and feeding it to laboratory rats suchas Crl:CD(SD)IGS BR rats, at a concentration of about 100milligrams/Kilogram body weight/day for 90 days, and then sacrificingand evaluating the rats by various medical testing procedures to showthat the amide compound of Formula (I) causes no adverse toxic effectson the rats.

Four of the compounds (further described in the examples 1, 24, 26, and30 below) have been successfully evaluated in the FEMA-GRAS process,declared to be “Generally Recognized As Safe.”

The Compounds of the Invention as Savory Taste Enhancers

The amide compounds of Formula (I) and its various compound sub-genusesand species, as described above are intended to be savory tasteflavorant compounds or flavor modifiers for comestible or medicinalproducts. As is apparent from the teachings and Examples herein, manycompounds of Formula (I) are agonists of an hT1R1/hT1R3 “savory”receptor, at least at relatively high amide compound concentrations, andaccordingly many of the amide compounds of Formula (I) can have utilityas savory flavorants or flavor enhancers, at least at relatively highconcentrations.

Nevertheless, it is preferable to use as little of such artificialflavorants as possible, so as to minimize cost. Moreover, it isdesirable to develop comestible compositions that do not have any“off-tastes” that may result if the compounds described above are usedat unnecessarily high concentrations. Accordingly, it is desirable totest the compounds of Formula (I) for their effectiveness as tastereceptor agonists at lower concentration levels, so as to identify thebest and most effective amide compounds within the compounds of Formula(I). As was disclosed in WO 03/001876, and U.S. Patent Publication No.US 2003-0232407 A1, and as described hereinbelow, laboratory proceduresnow exist for measuring the agonist activities of compounds forhT1R1/hT1R3 “savory” receptors. Such measurement methods typicallymeasure an “EC₅₀”, i.e. the concentration at which the compound causes50% activation of the relevant receptor.

Preferably, the amide compounds of Formula (I) that are savory flavormodifiers have an EC50 for the hT1R1/hT1R3 receptor of less than about10 μM. More preferably, such amide compounds have an EC₅₀ for thehT1R1/hT1R3 receptor of less than about 5 μM, 3 μM, 2 μM, 1 μM, or 0.5μM.

In some embodiments, the amide compounds of Formula (I) are savoryflavor modulators or enhancers of the agonist activity of monosodiumglutamate for an hTIR1/hT1R3 receptor. Hereinbelow is described an assayprocedure for so-called EC₅₀ ratios, i.e. for dissolving a compound ofFormula (I) in water containing MSG, and measuring the degree to whichthe amide compound lowers the amount of MSG required to activate 50% ofthe available hT1R1/hT1R3 receptors. Preferably, the amide compounds ofFormula (I), when dissolved in a water solution comprising about 1 μM ofthe amide compound will decrease the observed EC₅₀ of monosodiumglutamate for an hT1R1/hT1R3 receptor expressed in an HEK293-Gα15 cellline by at least 50%, i.e. the amide compound will have an EC50 ratio ofat least 2.0, or preferably 3.0, 5.0, or 7.0.

The above identified assays are useful in identifying the most potent ofthe amide compounds of Formula (I) for savory taste modifier or enhancerproperties, and the results of such assays are believed to correlatewell with actual savory taste perception in animals and humans, butultimately the results of the assays can be confirmed, at least for themost potent of the compounds of Formula (I), by human taste testing.Such human taste testing experiments can be well quantified andcontrolled by tasting the candidate compounds in aqueous solutions, ascompared to control aqueous solution, or alternatively by tasting theamides of the inventions in actual food compositions.

Accordingly, in order to identify the more potent of the savory tastemodifiers or agents, a water solution comprising a savory flavormodifying amount of the amide compound should have a savory taste asjudged by the majority of a panel of at least eight human taste testers.

Correspondingly, in order to identify the more potent of the savorytaste enhancers, a water solution, comprising a savory flavor modifyingamount of an amide compound of Formula (I) and 12 mM monosodiumglutamate, would have an increased savory taste as compared to a controlwater solution comprising 12 mM monosodium glutamate, as determined bythe majority of a panel of at least eight human taste testers.Preferably, in order to identify the more potent of the savory tasteenhancers, a water solution comprising a savory flavor modifying amount(preferably about 30, 10, 5, or 2 ppm) of the amide compound of Formula(I) and 12 mM monosodium glutamate will have an increased savory tasteas compared to a control water solution comprising 12 mM monosodiumglutamate and 100 μM inosine monophosphate, as determined by themajority of a panel of at least eight human taste testers.

Moreover, as described hereinbelow, human taste testors have tastetested several of the amide and/or oxalamide compounds described aboveas formulated in model comestible formulations, and have typicallyreported that the modified comestible compositions do have the “savory,Umami” taste and/or mouth-watering characteristics characteristic offoods containing MSG, particularly if the amide and/or oxalamidecompounds are well dispersed in the comestible composition, atconcentrations of from about 0.1 to about 3 ppm, or from about 0.2 toabout 2 ppm. Accordingly, when properly formulated and applied toappropriate comestible food and drinks novel, and favorable uses for thecompounds described above include but are not limited to:

-   -   i. Intensified savory, meaty, and/or salty perceptions in flavor        concentrate ingredients such as bases, stocks, seasoning mixes,        autolyzed yeast extracts, and hydrolyzed vegetable proteins    -   ii. Intensified and enhanced spice intensities in flavoring        ingredients such as whole/ground/oleoresin extracts of various        herbs and spices,    -   iii. Increased flavor intensities of food acids such as acetic,        citric, malic, tartaric, phosphoric acids, and    -   iv. A novel savory dimensional flavor that intensifies kokumi        flavor and the flavors of other hydrolyzed protein-based        flavorants. Kokami is a Japanese word for the intensity and        continuity of a savory flavor.

If however the amide and/or oxalamide compounds are not adequatelydispersed in the composition, or concentrations of those compounds aretoo high, the savory flavor can “linger” longer than the taste of MSG,and metallic side tastes and/or a physical sensation of tingling can beperceptible. Such “off-tastes” can sometimes be masked, but to ensureconsistency and homogeneity of the flavors, careful processing,formulation, and application of the compounds into diluted“user-friendly” flavor concentrate compositions, which can be easilyhandled and applied to traditional comestible compositions are among theinventions described herein. Such flavorant concentrate compositions canbe either liquid or solid, or be composed of hydrophilic or hydrophobicflavor diluent components.

The savory amide and/or oxalamide compounds of the invention typicallyhave at least some water solubility, so that for appropriate end useapplications, simply dissolving the savory amide and/or oxalamidecompounds in water can form an aqueous solution of the savory amideand/or oxalamide compounds and/or MSG, AYE, or HVP, or other desirablecomponents. Nevertheless, higher concentrations of the savory amideand/or oxalamide compounds in savory flavorant concentrate compositionscan often be achieved by dissolving the compounds and other componentsin comestibly acceptable organic solvents. Accordingly, in some aspects,the inventions relate to processes for preparing a liquid savoryflavorant concentrate composition, comprising:

-   -   a) mixing in any order        -   i) a liquid phase comprising one or more comestibly            acceptable solvents selected from the group consisting of            benzyl alcohol, triethyl citrate, benzyl benzoate,            triacetin, glycerin, propylene glycol or a methyl or ethyl            ether thereof or an acetate ester thereof,        -   ii) monosodhxm glutamate or glutamic acid, and        -   iii) one or more amide compounds having the formula

-   -   wherein    -   (1) A is a 5 or 6 membered aryl or heteroaryl ring,    -   (2) m is 0, 1, 2, 3 or 4,    -   (3) each R^(1′) is independently selected from the group        consisting of hydroxy!, NH₂, SH, halogen, and a C₁-C₄ organic        radical, and    -   (4) R² has 3 to 15 carbon atoms and is a branched alkyl, a        cycloalkyl ring, or a heterocyclic ring, optionally substituted        with 1, 2, 3, or 4 substituent groups independently selected        from the group consisting of hydroxyl, NH₂, SH, halogen, and a        C₁-C₄ organic radical, or one or more comestibly acceptable        salts thereof, to form a liquid savory flavorant concentrate        composition comprising at least about 10 ppm of the one or more        amide compounds or a comestibly acceptable salt thereof.

In related aspects, the inventions relate to processes for preparing aliquid savory flavorant concentrate composition, comprising:

-   -   a) mixing in any order        -   i) a liquid phase comprising one or more comestibly            acceptable solvents selected from the group consisting of            benzyl alcohol, triethyl citrate, benzyl benzoate,            triacetin, glycerin, propylene glycol or a methyl or ethyl            ether thereof or an acetate ester thereof,        -   ii) monosodium glutamate or glutamic acid, and one or more            oxalamide compounds having the formula

-   -   wherein    -   (1) A and B are independently an aryl, heteroaryl, cycloalkyl,        or heterocycle comprising 5 to 12 ring atoms,    -   (2) m and n are independently 0, 1, 2, 3 or 4-8,    -   (3) R⁷⁰ and R⁸⁰ are independently selected from the group        consisting of hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH, SR⁹,        halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰, NR⁹COR¹⁰, SOR⁹,        SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl, cycloalkyl, cycloalkenyl,        aryl, heteroaryl, and heterocycle, and R⁹ and R¹⁰ are        independently selected from H, C₁-C₆ alkyl, C₃-C₆ cycloalkyl,        and C₁-C₆ alkenyl, or two of R⁷⁰ together form a methylenedioxy        ring,    -   or one or more comestibly acceptable salts thereof;    -   to form a liquid savory flavorant concentrate composition        comprising at least about 10 ppm of the one or more oxalamide        compounds or a comestibly acceptable salt thereof.

In many aspects, such liquid savory flavorant concentrate compositionscan comprise higher concentrations of from about 10 ppm to about 10,000ppm, from about 50 ppm to about 5,000 ppm, or from about 100 ppm toabout 1000 ppm of the one or more amide or oxalamide compounds, or acomestibly acceptable salt thereof, and the molar ratio of the sum ofthe moles of the monosodium glutamate or glutamic acid divided by thesum of the moles of the one or more amide compounds is from about 5:1 toabout 1000:1, or from about 10:1 to about 500:1, or from about 20:1 toabout 300:1. In many aspects, the liquid savory flavorant concentratecomposition further comprises water.

Moreover, as noted above, it has been unexpectedly discovered that infavorable applications involving liquid and/or semi-solid comestiblecompositions, the presence of the savory amide and/or oxalamidecompounds described above can notably intensify the human perception ofsalt (sodium chloride), so as to enable the production of reduced sodiumformulations of comestible compositions. Moreover, previously knownreduced sodium formulations of comestible compositions substitutepotassium chloride (KCl) for sodium chloride (NaCl), but the potassiumchloride can, if used in increasing concentrations, introduce a metallicoff-taste, an effect which Applicants have surprising discovered thatthe amide and/or oxalamide compounds can partially or completely mask.Accordingly, in some aspects, the inventions relate to a process forreducing sodium content in a savory soup, broth, bullion, sauce, orgravy comprising sodium chloride and monosodium glutamate by

-   -   a) reformulating an existing soup, broth, bullion, sauce, gravy        or a precursor thereof to comprise at least about 0.01 ppm of an        amide compound having the formula

-   -   wherein        -   i) A is a 5 or 6 membered aryl or heteroaryl ring,        -   ii) m is 0, 1, 2, 3 or 4,        -   iii) each R¹ is independently selected from the group            consisting of hydroxyl, NH₂, SH, halogen, and a C₁-C₄            organic radical, and        -   iv) R² has 3 to 15 carbon atoms and is a branched alkyl, a            cycloalkyl ring, or a heterocyclic ring, optionally            substituted with 1, 2, 3, or 4 substituent groups            independently selected from the group consisting of            hydroxyl, NH2, SH, halogen, and a C₁-C₄ organic radical,    -   or one or more comestibly acceptable salts thereof,    -   b) and reducing the amount of one or more sodium salts added to        the reformulated soup, broth, bullion, sauce, or gravy by at        least about 5% as compared to the existing soup, broth, bullion,        sauce, or gravy.

In related aspects, the inventions relate to a process for reducingsodium content in a savory soup, broth, bullion, sauce, or gravycomprising sodium chloride and monosodium glutamate by

-   -   i) reformulating an existing soup, broth, bullion, sauce, gravy        or a precursor thereof to comprise at least about 0.01 ppm of        one or more oxalamide compounds having the formula

-   -   wherein        -   (1) A and B are independently an aryl, heteroaryl,            cycloalkyl, or heterocycle comprising 5 to 12 ring atoms,        -   (2) m and n are independently 0, 1, 2, 3 or 4-8,        -   (3) R⁷⁰ and R⁸⁰ are independently selected from the group            consisting of hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH,            SR⁹, halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰,            NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,            cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle,            and R⁹ and R¹⁰ are independently selected from H, C₁-C₆            alkyl, C₃-C₆ cycloalkyl, and C₁-C₆ alkenyl, or two of R⁷⁰            together form a methylenedioxy ring,    -   or one or more comestibly acceptable salts thereof;    -   b) and reducing the amount of one or more sodium salts added to        the reformulated soup, broth, bouillon, sauce, or gravy by at        least about 5% as compared to the existing soup, broth, bullion,        sauce, or gravy.

In such reformulation processes, the amount of sodium added to the soup,broth, bullion, sauce, or gravy comprising the amide compound can bereduced by at least about 10, or optionally 15, 20, 25, 30, 35, 40, 45,or 50% by weight, as compared to previously existing soup, broth,bullion, sauce, or gravy formulations, yet maintain good taste asperceived by humans. Ideally, the salt content of the reformulated soup,broth, bullion, sauce, or gravy is indistinguishable by taste from thatof the previously existing soup, broth, bullion, sauce, or gravy asjudged by a majority of a panel of at least eight human taste testers.

In other aspects the inventions relate to a process for preparing asolid flavorant concentrate composition, comprising:

-   -   a) providing one or more amide compounds having the formula

-   -   wherein        -   i) A is a 5 or 6 membered aryl or heteroaryl ring,        -   ii) m is 0, 1, 2, 3 or 4,        -   iii) each R¹ is independently selected from the group            consisting of hydroxyl, NH₂, SH, halogen, and a C₁-C₄            organic radical, and        -   iv) R² has 3 to 15 carbon atoms and is a branched alkyl, a            cycloalkyl ring, or a heterocyclic ring, optionally            substituted with 1, 2, 3, or 4 substituent groups            independently selected from the group consisting of            hydroxyl, NH₂, SH, halogen, and a C₁-C₄ organic radical,    -   or one or more comestibly acceptable salts thereof, and    -   b) dissolving the one or more amide compounds or comestibly        acceptable salts thereof in one or more comestibly acceptable        liquids to form a flavorant solution;    -   c) contacting the flavorant solution with one or more comestibly        acceptable solid carriers or a solution, dispersion, or emulsion        thereof, to form an intermediate composition; and    -   d) removing or permitting the loss of liquids from the        intermediate composition so as to form a solid flavorant        concentrate composition.

Similar aspects of the inventions relate to a process for preparing asolid flavorant concentrate composition, comprising:

-   -   a) providing one or more oxalamide compounds having the formula

-   -   wherein        -   i) A and B are independently an aryl, heteroaryl,            cycloalkyl, or heterocycle comprising 5 to 12 ring atoms,        -   ii) m and n are independently 0, 1, 2, 3 or 4-8,        -   iii) R⁷⁰ and R⁸⁰ are independently selected from the group            consisting of hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH,            SR⁹, halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰,            NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,            cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle,            and R⁹ and R¹⁰ are independently selected from H, C₁-C₆            alkyl, C₃-C₆ cycloalkyl, and C₁-C₆ alkenyl, or two of R⁷⁰            together form a methylenedioxy ring, or one or more            comestibly acceptable salts thereof;    -   b) dissolving the one or more oxalamide compounds or comestibly        acceptable salts thereof in one or more comestibly acceptable        liquids to form a flavorant solution;    -   c) contacting the flavorant solution with one or more comestibly        acceptable solid carriers or a solution, dispersion, or emulsion        thereof, to form an intermediate composition; and    -   d) removing or permitting the evaporation of liquids from the        intermediate composition so as to form a solid flavorant        concentrate composition.

In such processes, the amide or oxalamide compound having a highlypotent flavoring effect is first diluted by dissolving it in one or morecomestibly acceptable liquids to form a flavorant solution wherein theflavorant molecules are dispersed or dissolved to lower and homogeneousconcentrations. Useful comestibly acceptable liquids for dispersing ordissolving the compounds include but are not limited to water, ethanol,propylene glycol, glycerin, triacetin, edible fats or oils comestiblyacceptable glyceride triesters, benzyl alcohol, triethyl citrate, andbenzyl benzoate.

The flavorant solution is then contacted with one or more comestiblyacceptable solid carriers or a solution, dispersion, or emulsionthereof, to form an intermediate composition that can be optionallyprocessed further to insure uniform distribution of the Umami compoundsin the intermediate composition. Suitable solid carriers includingedible polysaccharides such as natural or modified starches, vegetableflours, maltodextrin, gelatin type A, gelatin type B, polyphosphate,alginate, chitosan, carrageenan, pectin, starch, gum arabic,alfa-lactalbumin, beta-lactoglobumin, ovalbumin, polysorbitol,cyclodextrin, cellulose, methyl cellulose, ethyl cellulose,hydropropylmethylcellulose, carboxymethylcellulose, powdered milk, milkprotein, whey protein, soy protein, canola protein, albumin, koshergelatin, non-kosher gelatin, Halal gelatin, and non-Halal gelatin.

The further processing of the intermediate composition can comprisesimple mixing processes or more complex and effective processes such asmilling or homogenization. Any homogenization technique or apparatusknown in the art can be used, and many suitable homogenizers arecommercially available. Homogenization can involve the use ofsonication, pressure, and/or mechanical devices to homogenize a fluid.For example, the homogenization can be a two-step or two-stagehomogenization, a high pressure homogenization, a very-high pressurehomogenization, a rotator-stator homogenization, a blade homogenization,and the like.

In some aspects, a homogenization step can be a pressure-basedhomogenization technique operating at pressures of from about 500 toabout 12,000 psi, from about 1,000 to about 9,000 psi, or from about3,000 to about 6,000 psi. In still other examples, the homogenizationstep can be performed at about 500, 1000, 1500, 2000, 2500, 3000, 3500,4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500,10000, 10500, 11000, 11500, or 12000 psi, where any of the stated valuescan form an upper or lower endpoint when appropriate. Homogenization canbe used to create uniform and/or smaller particle sizes, down to theorder a micron or less.

After homogenization, the intermediate compositions can undergo furtherprocessing, such as being sterilized or pasteurized in solution,emulsion, or fluid dispersion form. Additional ingredients can also beadded, such as monosodium glutamate, inosine monophosphate, guanosinemonophosphate, autolyzed yeast extracts, hydrolyzed vegetable proteins,spices, stabilizers, buffers, anti-oxidants, and other conventional foodadditives and flavorings.

In the processes discussed above, after the intermediate composition hasbeen processed to insure uniform dispersion of the amide or oxalamidecompounds, removal of or loss of liquids from the intermediatecomposition is permitted, so as to form a solid flavorant concentratecomposition that will be employed to flavor final comestiblecompositions. Such loss of liquids can be induced by heating orevaporation, or active removal of liquids by well known processes suchas spray drying, to form the final solid flavorant concentratecomposition. It must be noted however that the solid flavorantconcentrate composition can retain some of the liquids (such as smallamounts of water, fats, or oils) even when in “solid” form.

In many aspects of the invention, the one or more amide compounds arepresent in the solid flavorant concentrate composition in an amount offrom about 100 to about 100,000 ppm, or alternatively 200 to 50,000, 500to 30,000, 700 to 20,000, or from 1000 to about 10,000 ppm.

Special processing issues can be encountered with attempting to preparelipophillic flavorant concentrate compositions comprising comestiblyacceptable fats or oils, in which the amide and/or oxalamide compoundshave limited solubility, and hence may be present in a solid form whichis not easily dispersed. One solution to this problem is to mill orhomogenize a mixture of the fats or oils and the particles of the amideor oxalamide compounds, so as to form a dispersion of microparticles.Accordingly, in some aspects, the inventions herein relate to a processfor preparing a lipophillic savory flavorant concentrate composition,comprising:

-   -   a) contacting one or more comestibly acceptable fats or oils,        and one or more amide compounds having the formula

-   -   wherein        -   i) A is a 5 or 6 membered aryl or heteroaryl ring,        -   ii) m is 0, 1, 2, 3 or 4,        -   iii) each R¹ is independently selected from the group            consisting of hydroxyl, NH₂, SH, halogen, and a C₁-C₄            organic radical, and        -   iv) R² has 3 to 15 carbon atoms and is a branched alkyl, a            cycloalkyl ring, or a heterocyclic ring, optionally            substituted with 1, 2, 3, or 4 substituent groups            independently selected from the group consisting of            hydroxyl, NH₂, SH, halogen, and a C₁-C₄ organic radical,        -   or one or more comestibly acceptable salts thereof, to form            a precursor flavorant mixture, and    -   b) processing the precursor flavorant mixture to form a        lipophillic savory flavorant concentrate composition wherein at        least a major portion of the one or more amide compounds or        comestibly acceptable salt thereof are present in the form of        dispersed and undissolved microparticles.

In related aspects, the inventions relate to a process for preparing alipophillic savory flavorant concentrate composition, comprising:

-   -   a) contacting one or more comestibly acceptable fats or oils,        and one or more oxalamide compounds having the formula

-   -   wherein        -   i) A and B are independently an aryl, heteroaryl,            cycloalkyl, or heterocycle comprising 5 to 12 ring atoms,        -   ii) m and n are independently 0, 1, 2, 3 or 4-8,        -   iii) R⁷⁰ and R⁸⁰ are independently selected from the group            consisting of hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH,            SR⁹, halogen, CN, NO₂, CO₂R⁹, COR⁹, CONR⁹R¹⁰, NR⁹R¹⁰,            NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,            cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle,            and R⁹ and R¹⁰ are independently selected from H, C₁-C₆            alkyl, C₃-C₆ cycloalkyl, and C₁₋C₆ alkenyl, or two of R⁷⁰            together form a methylenedioxy ring,        -   or one or more comestibly acceptable salts thereof; to form            a precursor flavorant mixture, and    -   b) processing the precursor flavorant mixture to form a        lipophillic savory flavorant concentrate composition wherein at        least a major portion of the one or more amide compounds or        comestibly acceptable salt thereof are present in the form of        dispersed and undissolved microparticles.

In such processes, the processing step, normally comprises a mechanicalstep that reduces the size of the particles of the amide or oxalamidecompound to desired size ranges of microparticles of the compounddispersed in the oil or fat. The desired size range of themicroparticles as dispersed in the oil or fat will vary with theintended application, but in many applications the desiredmicroparticles will have an average particle size of less than about100, 50, 40, 30, 20, 10, 5, 2, or 1 μm.

Mixtures of Savory Compounds

It has been found the various samide and oxalamide compounds disclosedherein, while typically having very high individual potency as savoryflavoring agrents, also have somewhat differing solubilities, rapidityin inducing and degrees of lingering savory flavors, side-tastes, etc.Applicants have surprisingly discovered that the savory flavoringeffect, or savory flavor enhancing effect, of the amide and/or oxalamidecompounds of the invention can be improved by using mixtures of one ortwo or more savory compounds of the invention when formulatingcompositions.

Accordingly, in some embodiments, the invention relates to comestiblecompositions comprising

-   -   a) a savory flavor modulating amount of one or more amide        compounds having the formula

-   -   wherein        -   i) A′ is a 5 or 6 membered aryl or heteroaryl ring,        -   ii) m is 0, 1, 2, 3 or 4,        -   iii) each R^(1′) is independently selected from the group            consisting of hydroxyl, NH₂, SH, halogen, and a C₁-C₄            organic radical, one or two of the R¹ substituent groups are            bonded together to form a saturated alkylenedioxy ring and        -   iv) R² has 3 to 15 carbon atoms and is a branched alkyl, a            cycloalkyl ring, or a heterocyclic ring, optionally            substituted with 1, 2, 3, or 4 substituent groups            independently selected from the group consisting of            hydroxyl, NH₂, SH, halogen, and a C_(I)-C_(I) organic            radical,        -   or one or more comestibly acceptable salts thereof; and    -   b) a savory flavor modulating amount of one or more oxalamide        compounds having the formula

-   -   wherein for the one or more oxalamide compounds        -   i) A and B are independently an aryl, heteroaryl,            cycloalkyl, or heterocycle comprising 5 to 12 ring atoms,        -   ii) m and n are independently 0, 1, 2, 3 or 4-8,        -   iii) R⁷⁰ and R⁸⁰ are independently selected from the group            consisting of hydrogen, alkyl, alkoxyl, alkoxy-alkyl, OH,            SR⁹, halogen, CN, NO₂, CO₂R⁹, COR^(S), CONR⁹R¹⁰, NR⁹R¹⁰,            NR⁹COR¹⁰, SOR⁹, SO₂R⁹, SO₂NR⁹R¹⁰, NR⁹SO₂R¹⁰, alkenyl,            cycloalkyl, cycloalkenyl, aryl, heteroaryl, and heterocycle,            and R⁹ and R¹⁰ are independently selected from H, C—C₆            alkyl, C₃-C₆ cycloalkyl, and C₁-C₆ alkenyl, or two of R⁷⁰            together form a methylenedioxy ring, or one or more            comestibly acceptable salts thereof.

In such compositions comprising both amide and oxalamide compounds, inmany embodiments the compositions further comprise monosodium glutamate(MSG). In such compositions, the combination of the amide and oxalamidecompounds can enhance the savory flavor of the MSG present, so that lessMSG and/or it's associated sodium ions are necessary to achieve thedesired level of savory flavoring effect, while providing for ahealthier comestible composition.

In such compositions comprising both amide and oxalamide compounds, thecomposition can comprise from about 0.1 to about 3 ppm, or from about0.2 to about 1 ppm of the one or more amide compounds, and from about0.1 to about 3 ppm, or from about 0.2 to about 1 ppm of the one or moreoxalamide compounds. In some embodiments, the total of the amide andoxalamide compounds is from about 0.2 to about 3 ppm, or from about 0.5to about 1.0 ppm. The amide and oxalamide compounds may be present inany molar or weight ratio, but in many embodiments the oxalamide toamide weight ratios are from about 1:1 to about 1:6, or from about 1:2to about 1:4.

The comestible compositions comprising the mixture of one or more amidecompounds and one or more oxalamide compounds can be any of the largevariety of classes, subclasses and species of comestible compositionsdescribed hereinbelow, but in some desirable embodiments, the comestiblecomposition is a bullion, broth, or soup; or a sauce or condiment; or avegetable or tomato juice, or a salad dressing or mayonnaise; or asavory seasoning composition, or a battered fried food.

The comestible compositions comprising the mixture of one or more amidecompounds and one or more oxalamide compounds can also be a flavorconcentrate composition. Such flavor concentrate compositions may or maynot comprise MSG, and can comprise from about 10 to about 10,000 ppm ofthe one or more amide or oxalamide compounds. Such flavor concentratecomposition can be a liquid composition, or a solid composition.

In some embodiments, the comestible compositions comprising the mixtureof one or more amide compounds and one or more oxalamide compoundscomprise an amide compound having the formula

and an oxalamide compound have the formula

Using the Compounds of Formula (I) to Prepare Comestible Compositions

Flavors, flavor modifiers, flavoring agents, flavor enhancers, savory(“umami”) flavoring agents and/or flavor enhancers, according to theinvention have application in foods, beverages and medicinalcompositions wherein savory compounds are conventionally utilized. Thesecompositions include compositions for human and animal consumption. Thisincludes foods for consumption by agricultural animals, pets and zooanimals.

Those of ordinary skill in the art of preparing and selling comestiblecompositions (i.e. edible foods or beverages, or precursors or flavormodifiers thereof) are well aware of a large variety of classes,subclasses and species of the comestible compositions, and utilizewell-known and recognized terms of art to refer to those comestiblecompositions while endeavoring to prepare and sell various of thosecompositions. Such a list of terms of art is enumerated below, and it isspecifically contemplated hereby that the various subgenuses and speciesof the compounds of Formula (I) could be used to modify or enhance thesavory of the following list comestible compositions, either singly orin all reasonable combinations or mixtures thereof:

-   -   Bread, pastries, cakes, packaged/industrial cakes,        unpackaged/artisanal cakes, cookies, chocolate coated biscuits,        sandwich biscuits, filled biscuits, savoury biscuits and        crackers, bread substitutes, breakfast cereals, rte cereals,        family breakfast cereals, flakes, muesli, other rte cereals,        children's breakfast cereals, hot cereals, dairy products, milk,        fresh/pasteurised milk, full fat fresh/pasteurised milk, semi        skimmed fresh/pasteurised milk, long-life/uht milk, full fat        long life/uht milk, semi skimmed long life/uht milk, fat-free        long life/uht milk, goat milk, condensed/evaporated milk, plain        condensed/evaporated milk, flavoured, functional and other        condensed milk, flavoured milk drinks, dairy only flavoured milk        drinks, flavoured milk drinks with fruit juice, soy milk, sour        milk drinks, fermented dairy drinks, coffee whiteners, powder        milk, flavoured powder milk drinks, cream, cheese, processed        cheese, spreadable processed cheese, uπspreadable processed        cheese, unprocessed cheese, spreadable unprocessed cheese, hard        cheese, packaged hard cheese, unpackaged hard cheese, yoghurt,        plain/natural yoghurt, flavoured yoghurt, fruited yoghurt,        probiotic yoghurt, drinking yoghurt, regular drinking yoghurt,        probiotic drinking yoghurt, chilled snacks, fromage frais and        quark, plain fromage frais and quark, flavoured fromage frais        and quark, savoury fromage frais and quark, sweet and savoury        snacks, fruit snacks, chips/crisps, extruded snacks,        tortilla/corn chips, popcorn, pretzels, nuts, other sweet and        savoury snacks, snack bars, granola bars, breakfast bars, energy        bars, fruit bars, other snack bars, meal replacement products,        slimming products, convalescence drinks, ready meals, canned        ready meals, frozen ready meals, dried ready meals, chilled        ready meals, dinner mixes, frozen pizza, chilled pizza, soup,        canned soup, dehydrated soup, instant soup, chilled soup, uht        soup, frozen soup, pasta, canned pasta, dried pasta,        chilled/fresh pasta, noodles, plain noodles, instant noodles,        cups/bowl instant noodles, pouch instant noodles, chilled        noodles, snack noodles, canned food, canned meat and meat        products, canned fish/seafood, canned vegetables, canned        tomatoes, canned beans, canned fruit, canned ready meals, canned        soup, canned pasta, other canned foods, frozen food, frozen        processed red meat, frozen processed poultry, frozen processed        fish/seafood, frozen processed vegetables, frozen meat        substitutes, frozen potatoes, oven baked potato chips, other        oven baked potato products, non-oven frozen potatoes, frozen        bakery products, frozen desserts, frozen ready meals, frozen        pizza, frozen soup, frozen noodles, other frozen food, dried        food, dessert mixes, dried ready meals, dehydrated soup, instant        soup, dried pasta, plain noodles, instant noodles, cups/bowl        instant noodles, pouch instant noodles, chilled food, chilled        processed meats, chilled fish/seafood products, chilled        processed fish, chilled coated fish, chilled smoked fish,        chilled lunch kit, chilled ready meals, chilled pizza, chilled        soup, chilled/fresh pasta, chilled noodles, oils and fats, olive        oil, vegetable and seed oil, cooking fats, butter, margarine,        spreadable oils and fats, functional spreadable oils and fats,        sauces, dressings and condiments, tomato pastes and purees,        bouillon/stock cubes, stock cubes, gravy granules, liquid stocks        and fonds, herbs and spices, fermented sauces, soy based sauces,        pasta sauces, wet sauces, dry sauces/powder mixes, ketchup,        mayonnaise, regular mayonnaise, mustard, salad dressings,        regular salad dressings, low fat salad dressings, vinaigrettes,        dips, pickled products, other sauces, dressings and condiments,        baby food, milk formula, standard milk formula, follow-on milk        formula, toddler milk formula, hypoallergenic milk formula,        prepared baby food, dried baby food, other baby food, spreads,        jams and preserves, honey, chocolate spreads, nut-based spreads,        and yeast-based spreads.

Preferably, the compounds of Formula (I) can be used to modify orenhance the savory flavor of one or more of the following sub-genuses ofcomestible compositions: confectioneries, bakery products, dairyproducts, sweet and savory snacks, snack bars, meal replacementproducts, ready meals, soups, pastas, noodles, canned foods, frozenfoods, dried foods, chilled foods, oils and fats, baby foods, orspreads, or a mixture thereof. Among the more favored sub-genera of foodcompositions are the comestible compositions listed in the followingtable:

Product Usage Sensory and flavor attributes Bouillon, Broths, Soups,Umami flavor, MSG flavor characteristics Ramen Noodles and enhancementof savory, brothy and meat flavors. Low Sodium Broth, Soups Umamiflavor, enhancement of savory, brothy and meat flavors; diminishment ofKC1 off-tastes such as bitter and metallic notes. Savory sauces such astomato Intensified savory, spice flavors, cheese based, gravy, cheeses,soy flavors; umami and MSG flavor sauce based, condimentscharacteristics; blending and rounding fo all flavors. Vegetable andtomato juices Savory, umami, mouth-watering flavor enhancements, reducedtartness Salad Dressings and Cheese or ranch type flavors, acid flavorMayonnaise is diminished and savory flavor blends. Mayonnaise and highacid type dressings and the acid flavor is intensified along withsavory, umami flavor qualities. Side dishes: Battered French Umami,savory and mouth-watering fries, fried appetizers, rice flavor.Intensified spice and savory and potato sides flavors. Rounded flavorcharacteristics, increased perceptions of salt or sodium. Savory topicalseasonings for Umami, savory and mouth-watering snack foods and Frenchfries, flavors. Intensified spices and savory etc. flavors. Roundedflavor characteristics. Meat, Poultry, Seafood Umami, savory andmouth-watering seasonings and marinades flavors. Intensified spices andsavory flavors. Rounded flavor characteristics.

In general an ingestible composition will be produced that contains asufficient amount of at least one compound within the scope of Formula(I) or its various subgenuses described hereinabove to produce acomposition having the desired flavor or taste characteristics such as“savory” taste characteristics.

Typically at least a savory flavor modulating amount, a savory flavoringagent amount, of one or more of the compounds of Formula (I), or thesavory flavorant concentrate compositions described herein will be addedto the comestible or medicinal product, so that the savory modifiedcomestible or medicinal product has an increased savory and/or sweettaste as compared to the comestible or medicinal product preparedwithout the amide compound, as judged by human beings or animals ingeneral, or in the case of formulations testing, as judged by a majorityof a panel of at least eight human taste testers, via proceduresdescribed elsewhere herein.

The concentration of savory or sweet flavoring agent needed to modulateor improve the flavor of the comestible or medicinal product orcomposition will of course vary dependent on many variables, includingthe specific type of ingestible composition, what savory compounds arepresent and the concentrations thereof, and the effect of the particularcompound on such savory compounds. As noted, a significant applicationof the compounds of Formula (I) is for modulating (inducing, enhancingor inhibiting) the savory or sweet tastes or other taste properties ofother natural or synthetic savory tastants. A broad but also low rangeof concentrations of the amide compounds of Formula (I) would typicallybe required, i.e. from about 0.001 ppm to 100 ppm, or narroweralternative ranges from about 0.1 ppm to about 10 ppm, from about 0.01ppm to about 30 ppm, from about 0.05 ppm to about 15 ppm, from about 0.1ppm to about 5 ppm, or from about 0.1 ppm to about 3 ppm.

Examples of foods and beverages wherein compounds according to theinvention may be incorporated include by way of example the Wet SoupCategory, the Dehydrated and Culinary Food Category, the BeverageCategory, the Frozen Food Category, the Snack Food Category, andseasonings or seasoning blends.

“Wet Soup Category” means wet/liquid soups regardless of concentrationor container, including frozen Soups. For the purpose of this definitionsoup(s) means a food prepared from meat, poultry, fish, vegetables,grains, fruit and other ingredients, cooked in a liquid which mayinclude visible pieces of some or all of these ingredients. It may beclear (as a broth) or thick (as a chowder), smooth, pureed or chunky,ready-to-serve, semi-condensed or condensed and may be served hot orcold, as a first course or as the main course of a meal or as a betweenmeal snack (sipped like a beverage). Soup may be used as an ingredientfor preparing other meal components and may range from broths (consommé)to sauces (cream or cheese-based soups).

“Dehydrated and Culinary Food Category” means: (i) Cooking aid productssuch as: powders, granules, pastes, concentrated liquid products,including concentrated bouillon, bouillon and bouillon like products inpressed cubes, tablets or powder or granulated form, which are soldseparately as a finished product or as an ingredient within a product,sauces and recipe mixes (regardless of technology); (ii) Meal solutionsproducts such as: dehydrated and freeze dried soups, includingdehydrated soup mixes, dehydrated instant soups, dehydratedready-to-cook soups, dehydrated or ambient preparations of ready-madedishes, meals and single serve entrees including pasta, potato and ricedishes; and (iii) Meal embellishment products such as: condiments,marinades, salad dressings, salad toppings, dips, breading, battermixes, shelf stable spreads, barbecue sauces, liquid recipe mixes,concentrates, sauces or sauce mixes, including recipe mixes for salad,sold as a finished product or as an ingredient within a product, whetherdehydrated, liquid or frozen.

“Beverage Category” means beverages, beverage mixes and concentrates,including but not limited to, alcoholic and non-alcoholic ready to drinkand dry powdered beverages.

Other examples of foods and beverages wherein compounds according to theinvention may be incorporated included by way of example carbonated andnon-carbonated beverages, e.g., fruit or vegetable juices, alcoholic andnon-alcoholic beverages, confectionary products, e.g., salad dressings,and other condiments, cereal, and other breakfast foods, canned fruitsand fruit sauces and the like.

Additionally, the subject compounds can be used in flavor preparationsto be added to foods and beverages. In preferred instances thecomposition will comprise another flavor or taste modifier such as asavory tastant.

The amide compounds of Formula (I) and its various subgenuses can becombined with or applied to the comestible or medicinal products orprecursor thereof in any of innumerable ways known to cooks the worldover, or producers of comestible or medicinal products. For example, theamide compounds of Formula (I) could be dissolved in or dispersed in orone one of many comestibly acceptable liquids, solids, or othercarriers, such as water at neutral, acidic, or basic pH, fruit orvegetable juices, vinegar, marinades, beer, wine, natural water/fatemulsions such as milk or condensed milk, edible oils and shortenings,fatty acids, certain low molecular weight oligomers of propylene glycol,glyceryl esters of fatty acids, and dispersions or emulsions of suchhydrophobic substances in aqueous media, salts such as sodium chloride,vegetable flours, solvents such as ethanol, solid edible diluents suchas vegetable powders or flours, and the like, and then combined withprecursors of the comestible or medicinal products, or applied directlyto the comestible or medicinal products.

Other techniques of food preparation and/or flavorant application can beused to formulate the compounds of the invention to prepare comestiblecomposition. In “dry blending” particles the high potency savorycompounds of the inventions, or solid flavor concentrate compositionsthereof are simply mixed as solids with other flavoring ingredientsand/or carriers or diluents, to hopefully produce a homogenous flavorpowder. The carriers or diluents are well known in the art, such asmaltodextrin, modified food starch, various hydrocolloid gums such asgum acacia, gum Arabic, etc, or salt or sugar can be used alone or incombination. Properly formulated dry blended compositions should notseparate, stratify, or segregate particles, which can cause flavorinhomogeneity or inconsistency.

In another example, solid flavorant concentrate compositions can beprepared by a process of agglomeration, which can also be referred to asfluid bed processing. In this process the compounds of the invention orconcentrate compositions thereof are spray coated as solid particlesonto diluent or carrier core material particles (such as ediblepolysaccharides, starches, etc) suspended in a column of moving air atcontrolled temperatures and humidity. The movement of the particles fromthe bottom of the chamber through the aerosol to the top of the chamberis random and produces a uniform coating of the compounds of theinvention on the core material. The particles coalesce to form porousagglomerates. The finished product is removed from the chamber, andoften put through a final drying step and cooling procedure prior topackaging. The final agglomerate material is a matrix of pores,crystallization, coagulation and or polymerization. The resulting coarseparticles can induce flavor effects such as flavor masking, varied timerelease of flavor or delayed flavor release to reduce any ‘umamilingering’ of the compounds described herein, and/or match the umamiflavor release to match the time/intensity and flavor release of MSG.

Another technique for modifying the flavor release characteristics ofthe compounds described herein is the technique of non-thermal flavorinfusion and complexing. In this technique, the compounds describedherein are processed so that they become complexed by a modified foodstarch, typically a beta cyclodextrin. The savory compound of theinvention is complexed by the ring structure of the modified starch, tomodify the rate and extent of flavor release or specific flavordelivery. Various methods can be used to complexing a cyclodextrin andthe compounds of the invention. Typically a high shear mixer or shakeris used to solubilize the compound of the inventions and thecyclodextring into an aqueous solution (typically 20-40% water) thenfiltering off the resulting precipitated complex. The resulting pastemay be used as is, or it can be dried and or ground to a powder form.The aqueous paste can be dried using conventional hot air ovens, spraydryers, vacuum dryers, freeze dryers and or agglomerators. Morefrequently employed methods of drying the agglomerates is freeze dryingor vacuum drying at comparatively lower drying temperatures of 140-185°F. An alternative method is to blend the solid cyclodextrin with flavormolecules in a kneading application. This forms a solid water/flavormolecule water paste. This also can be used or further dried and groundinto a powder form. This technology can reduce or increase the ‘umamilingering’ (dependent upon consumer liking and preference) and/orimprove the umami flavor release to match the time/intensity and flavorrelease of MSG, or to provide for controlled flavor delivery in variousfood formulations.

The liquid flavor concentrate compositions of the invention are oftenprepared using the technique of Liquid Flavor Compounding, whichinvolves attempting to “load” the flavor concentrate with as much of thesavory compounds as possible while reducing the water content as much aspossible in the liquid flavor concentrate composition.

Making the Amide Compounds of Formula (I)

The starting materials used in preparing the compounds of the invention,i.e. the various structural subclasses and species of the amidecompounds of Formula (I) and their synthetic precursors, as well asmethods for making the compounds described abover are disclosed in U.S.Patent Publication No. US 2005/0084506 A1 and U.S. Patent PublicationNo. US 2006/0045953 A1, incorporated herein by reference, or asdescribed below.

Synthetic Methods

The following Schemes and Examples are provided for the guidance of thereader, and represent a variety of methods for making the amidecompounds disclosed herein. The disclosed methods are exemplary only,not limiting, and be apparent to one or ordinary skill in the art thatother methods, many of which are known in the art, may be employed toprepare the amide compounds of the various embodiments of the invention.Such methods specifically include solid phase based chemistries,including combinatorial chemistry.

Amides are often prepared by the condensation of carboxylic acids and/ortheir derivatives (such as esters, acid halides etc) with primary orsecondary amines, often in the presence of dehydrating agents, couplingagents, and/or appropriate catalysts. Large numbers of suitable startingmaterials, such as primary and secondary amines, and carboxylic acidsand their derivatives, can be readily synthesized by methods known inthe literature or are readily available commercially. In some cases,methods for synthesis of certain amine or carboxylic acid startingmaterials are given below.

As shown in Scheme 1a, amide derivatives (I) can be prepared from thecoupling of acid derivatives (II) with amines (III), for example in thepresence of a coupling reagent such as1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride and a base.In Method A, a polymer supported (PS) carbodiimide is used. Method Buses a non-polymer supported carbodiimide.

As shown in Scheme 1b, amide derivatives (I) are alternatively preparedfrom the coupling of acid halides, esters, or anhydrides (IV) withamines (III) in the presence of a base.

For example, the compound of Example 1 shown below can be prepared andthen purified by the procedures shown in Examples 1 and 1-1.

In related aspects, the inventions disclosed herein include a processfor preparing the compound of Example 1, i.e.2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide having theformula:

The improved process provides several improvements over the originallaboratory procedure by taking advantage of a number of surprisingdiscoveries which will be discussed herein below in detail.

The process comprises:

-   -   a) reacting piperonylic acid having the formula:

with a reagent capable of reacting with the carboxylic acid to formingan acid chloride, such reagents include but are not limited to thionylchloride, oxalyl chloride, and phosphorous oxychloride to formpiperonoyl chloride having the formula:

and

-   -   b) reacting piperonoyl chloride formed in step (a) with        4-heptylamine having the formula:

-   -   to form        2-H-benzo[3,4-d]1,3-dioxolan-S-yl-N-(propylbutyl)-carboxamide.

Step (a)

Step (a) relates to the formation of piperonoyl chloride frompiperonylic acid. The piperonylic acid starting material is readilyavailable from manufacturers, for example, Alfa Aesar GmbH & Co. KG®,Alfa Aesar A Johnson Matthey Company®, and Alfa Aesar Johnson Mattheyplc®.

Step (a) utilizes a reagent capable of reacting with a carboxylic acidto form an activate carbonyl group in the form of an acid chloride.Non-limiting reagents with are suitable for this step include reagentscomprising thionyl chloride, oxalyl chloride, and phosphorousoxychloride, or mixtures thereof.

To catalyze the formation of the acid chloride a small or catalyticamount of a reagent such as dimethylformamide can be used to increasereaction rates and yield of the acid chloride.

The reaction may also be conducted in the presence of a solvent,non-limiting examples of which include a solvent chosen from methylenechloride, chloroform, and tetrahydrofuran.

In some embodiments, step (a) comprises one or more of the further stepsof:

-   -   i) combining piperonylic acid, methylene chloride and        dimethylformamide to form a liquid admixture;    -   ii) cooling the admixture to about 0° C. to form a cooled        admixture;    -   iii) adding said reagent capable of forming an acid chloride to        said cooled admixture at or below a temperature of about 10° C.        to form a reaction mixture; or    -   iv) heating to reflux said reaction mixture after the addition        of said acid chloride forming reagent to form a solution of        piperonoyl chloride.

Step (a)(i) relates to the use of methylene chloride as a solvent andthe additional use of dimethylformamide as a catalyst for the formationof piperonoyl chloride.

Step (a)(ii) relates to the reaction step wherein the admixture formedin step (a)(i) is pre-cooled to about 0° C. prior to the addition of theacid chloride forming reagent.

Step (a)(iii) relates to the iteration wherein the acid chloride formingreagent is thionyl chloride and it is added at or below a temperature ofabout 10° C.

Step (a)(iv) relates to the fact the formulator also has the choice ofheating, to reflux the reaction mixture formed in step (a)(iii) afterthe addition of thionyl chloride to insure complete formation ofpiperonoyl chloride.

Step (a) may also be conducted in the presence of an organic base asdescribed herein above for the purpose of acting as an acid sponge. Manytertiary amines are suitable, and triethylamine is a readily available,inexpensive, and safely utilized organic base which has been found to becompatible with the process of the present invention.

Step (b)

Step (b) relates to the formation of the final product2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide, andcomprises reacting piperonoyl chloride formed in step (a) with4-heptylamine. 4-aminoheptane is readily available commercially.

In some embodiments, step (b) comprises:

-   -   i) combining 4-heptylamine, triethylamine, methylene chloride,        and dimethylformamide to form a solution of 4-heptylamine;    -   ii) adding to the piperonoyl chloride to said solution of        4-heptylamine at a temperature below about 5° C., to form a        reaction mixture; and    -   iii) warming the reaction mixture to a temperature from about        20° C. to about 25° C. to form a crude reaction solution        comprising        2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide.

Step (b)(i) relates to the use of methylene chloride as a solvent andthe additional use of dimethylformamide as a catalyst for the formationof the final product.

Step (b)(ii) relates to one iteration of the reaction step whereinpiperonoyl chloride is added to the solution formed in step (b)(i) at atemperature below about 5° C.

Step (b)(iii) relates to the iteration wherein the formulator can warmthe solution, for example to a temperature from about 20° C. to about25° C. to insure completeness of the reaction.

However, the formulator can choose to add further steps to step (b), forexample,

-   -   iv) cooling the crude reaction solution obtained in step        (b)(iii) to a temperature of from about 0° C. to about 5° C. and        adding water to form a biphasic solution;    -   v) working up said biphasic solution by removing the aqueous        phase and treating the resulting organic phase with the        following solutions in the order of:        -   1. an aqueous solution of hydrochloric acid having a            normality of from about 0.1 N to about 2.0 N;        -   2. a saturated aqueous solution of sodium bicarbonate; and        -   3. a saturated aqueous solution of sodium chloride; to form            a solution of            2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide;    -   vi) removing the organic phase to form crude        2-H-benzo[3,4-d]1,3-dioxolan-5 yl-N-(propylbutyl)-carboxamide;        and    -   vii) forming a slurry of the crude        2-H-benzo[3,4-d]1.3-dioxolan-5-yl-N-(propylbutyl)-carboxamide        and isolating by filtration        2-H-benzo[3,4-d]1,3-dioxolan-5-yl-N-(propylbutyl)-carboxamide.

These workup steps will, depending upon the needs of the formulator,provide a procedure for isolation of the desired product.

As a general procedure, one amine is allowed to react with ethyl oxalylchloride in the presence of tertiary amine in organic solvent, such asdioxane, acetonitrile, tetrahydrofuran, tetrahydropyran, anddimethylformamide, at room temperature for 0.5-2 hours. Then the secondamine is added and the suspension is heated at 80° C. using oil bathovernight or at 160° C. in a microwave reactor for 5 minutes. Thereaction mixture can be subject to preparative HPLC, or an aqueouswork-up and the crude product can typically be readily purified byrecrystallization, flash column chromatography, or other methods wellknown to those of ordinary skill in the art to afford the pureoxalamide.

A very wide variety of carboxylic acid derivatives that are suitableprecursors of the R¹ groups of the amides of Formulas (I), and varioussubgenuses of the compounds of Formula (I) are readily available bymethods or ready adaptation of methods known in the prior art, or areavailable commercially. In particular, the substituted aryl orheteroaryl carboxylic acid compounds that are precursors of thecompounds of Formula (II) are often readily available commercially, orthrough use of very well known synthetic methodologies. Similarly, manyamine compounds that are suitable precursors of the amide compounds ofFormula (I) are readily available commercially or through known methodsof synthesis. Nevertheless, disclosed in the Schemes and/or Examplesbelow are methods for synthesizing certain starting building blockprecursors of the R¹, R², and R³ groups.

In some aspects, the inventions relate to improved methods ofsynthesizing the oxalamide compounds described above, and theirsynthetic precursors.

In some embodiments, the inventions disclosed herein relate to a processfor preparingN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide having theformula:

N-(2,4-Dimethoxyberizyl)-N[2-(pyridin-2-yl)ethyl]oxalamide is a newFEMA-GRAS approved high intensity savory compound that can substitutefor or significantly enhance the savory flavor of monosodium glutamate(MSG). The process disclosed here provides several improvements over theknown laboratory procedures by taking advantage of a number ofsurprising discoveries which will be discussed herein below in detail.

In some embodiments, the improved processes comprise:

-   -   a) condensing 2,4-dimethoxybenzylamine or a salt thereof with a        2-chloro-2-oxoacetate ester having the formula:

in the presence of a tertiary amine base and a solvent or solventmixture or aromatic solvents comprising one or more of toluene,o-xylene, m-xylene, p-xylene, or nitrobenzene, to form a solution of a2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester having the formula:

-   -   wherein R is C₁-C₄ linear or branched alkyl; and    -   b) reacting the solution of the        2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester formed in        step (a) with 2-(pyridin-2-yl)ethylamine having the formula:

-   -   to form        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide.

Step (a)

Step (a) relates to the formation of a2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester having the formula:

wherein R is C₁-C₄ linear or branched alkyl. The formulator dealing withlarge scale processes will appreciate that the large scale synthesis ofa molecule entails many considerations, as well as variables. Oneimportant factor is the availability of starting materials. The processof the present invention makes use of 2-chloro-2-oxoacetate estershaving the formula:

wherein R is a C₁-C₄ linear or branched alkyl group. This material maybe prepared by procedures well known to those of ordinary skill in theart by procedures from readily available starting materials, inter alia,oxalyl chloride and corresponding alcohols; methanol, ethanol,tert-butanol and the like. These reagents have the further advantagethat they can be prepared as needed or can be stored for later use.Example 1 herein below utilizes ethyl 2-chloro-2-oxoacetate.

2,4-Dimethoxybenzylamine is conveniently stored as a salt, for example,as an ammonium salt, inter alia, the hydrochloride and the hydrobromide,which (in contrast to the free amine form), are oxidatively stablecrystalline solids with long shelf life. In addition,2,4-dimethoxybenzylamine hydrochloride is readily available as astarting material, for example, from Fisher Scientific® (Catalogue No.AC17651-0050).

The choice of solvent, however, as in the case of the present invention,is important. As a general rule of thumb, for. every ten degrees rise inreaction temperature, the reaction rate is doubled. Although many otherfactors may mitigate the actual increase in reaction rate over aparticular temperature range, all things being equal, increasing thetemperature favors faster, more complete, and more productive chemicalreactions. The present invention discloses the surprising finding thataromatic solvent mixtures, which are high boiling and immiscible withwater can be used to provide higher rates and productivity, as well aseasy subsequent product processing by extraction. When the first step ofthe reaction is conducted in a solvent or solvent mixture comprising oneor more of toluene, o-xylene, m-xylene, p-xylene, nitrobenzene,especially toluene, the reaction through put is enhanced, and theresulting solutions can be readily processed downstream.

The choice of one of these solvents or mixtures of solvents alsoimproves the separation of the organic and aqueous phases if theformulator chooses to isolate and/or purify the product formed in step(a) by extraction. In particular, toluene can be obtained in high purityand at low cost from a wide range of suppliers and can also be used toazeotropically remove any water which may persist after any optionaldrying step.

Although the 2,4-dimethoxybenzylamine can be used as a free base, step(a) can be conducted in the presence of an organic base for severalpurposes, inter alia, to liberate the 2,4-dimethoxybenzylamine from itshydrochloride salt or to act as a sponge for any acid formed in thereaction. Any non-reactive tertiary organic base capable of coordinationof the liberated acid is acceptable. Trialkylamines, trialkoxyamines, orheteroaromatic amines are particularly suitable. Non-limiting examplesof trialkyl amines include triethylamine, diisopropylethylamine, andmethyldiisopropylamine. A non-limiting example of a trialkoxyamineincludes triethanolamine. Non-limiting examples of heteroaromatic aminesincludes pyridine and lutidine. Other non-nucleophilic amines, interalia, 1,8-diazabicyclo[5.4.0]undec-7-ene are also suitable for use inthe process of the present invention. Triethylamine is a readilyavailable, inexpensive, and safely utilized organic base which has beenfound to be compatible with the process of the present invention.

In one embodiment, the process for preparingN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide step (a)comprises:

-   -   a) reacting 2,4-dimethoxybenzylamine, or a salt thereof, with        ethyl 2-chloro-2-oxoacetate ester in the presence of        triethylamine and toluene, said step having one or more of the        further steps of:        -   i) combining 2,4-dimethoxybenzylamine, or an ammonium salt            thereof, triethylamine, and toluene to form an admixture;        -   ii) cooling said admixture to about 0° C. to form a cooled            admixture;        -   iii) adding to said cooled admixture, while maintaining the            temperature of said admixture at or below about 10° C.,            ethyl 2-chloro-2-oxoacetate ester to form a reaction            solution; and        -   iv) allowing said reaction solution to warm to from about            20° C. to about 27° C. to form impure ethyl            2-(2,4-dimethoxybenzylamino)-2-oxoacetate.

Step (a)(i) relates to the use of toluene as a solvent and the use oftriethylamine to serve as a source for removing the HCl which is formed,as well as to liberate the 2,4-dimethoxybenzyl free amine when anammonium salt is used as a starting material.

Step (a)(ii) relates to one iteration of the reaction step wherein theadmixture which is formed is cooled to about 0° C. prior to the additionof ethyl 2-chloro-2-oxoacetate ester, which in this iteration, asindicated in step (a)(iii) is added at a temperature at or below about10° C.

Step (a)(iv) relates to the final step of this iteration wherein thereaction solution which comprises the impure ethyl2-(2,4-dimethoxybenzylamino)-2-oxoacetate and any unreacted startingmaterials is allowed to warm to from about 20° C. to about 27° C. toensure reaction completion.

In a further iteration of Step (a), this step may also comprise one ormore of the additional steps of:

-   -   v) adding an aqueous solution of hydrochloric acid to the        solution of a 2-(2,4-dimethoxybenzylamino)-2-oxo acetate ester        to form an organic liquid phase comprising the        2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester and an aqueous        phase;    -   vi) drying said organic liquid phase comprising the        2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester to form a dry        solution of the 2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester.

Such an extraction procedure can be used to remove any water solubleimpurities or salts formed in the first reaction Step (a).

If desired or necessary, the intermediate can be fully purified beforereaction, the formulator may also choose to add the following proceduresto Step (a):

-   -   vii) removing the toluene from said dry solution of ethyl        2-(2,4-dimethoxybenzylamino)-2-oxoacetate in toluene to form a        solid ethyl 2-(2,4-dimethoxybenzylamino)-2-oxoacetate; and    -   viii) purifying said solid ethyl        2-(2,4-dimethoxybenzyl-amino)-2-oxoacetate.

The formulator will recognize that the addition of these steps willafford the user of the present process many options, one of which is tohave purified intermediate ethyl2-(2,4-dimethoxybenzyl-amino)-2-oxoacetate. This option is especiallyimportant because the final products of the present processes aretastants which are, in some aspects, anticipated for use in foodproducts consumed by humans. Therefore, the final step may necessarilybe required to be conducted in a facility different from the facilitywhere step (a) is performed. Having the intermediate in pure form allowsthe formulator to finish the process of the present invention in asecond facility or in segregated reactors.

Step (b)

Step (b) relates to the formation of the final productN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide comprisingreacting the 2-(2,4-dimethoxybenzylamino)-2-oxoacetate ester formed instep (a) with 2-(pyridin-2-yl)ethylamine having the formula:

Again this step can utilize the aforementioned solvents, for example,toluene if desired by the formulator. Once again the choice of steps bywhich the final product is formed takes advantage of the fact that2-(2-aminoethyl)pyridine is available commercially, for example, by ABCRGmbH & Co. KG, Chemos GmbH, Connect Marketing GmbH, and Rich FineChemicals Co., Ltd.

In additional embodiments of the process for preparingN-(2,4-dimethoxybenzyl-N′-[2-(pyridin-2-yl)ethyl]oxalamide relates tothe following, step (b) comprises one or more of:

-   -   i) mixing 2-(2-aminoethyl)pyridine to the solution of step (a)        to form a reaction solution, and    -   ii) heating said reaction solution to form        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide.

Step (b)(i) utilizes the solution obtained in step (a) withoutperforming any optional work-up steps.

Step (b)(ii) affords the formulator a temperature range option forconducting the reaction, for example, bringing the solution to reflux inthe higher boiling solvent toluene which is used as the solvent in step(a), thereby, increasing the reaction through put.

Accordingly, in some embodiments of Step (b), this step may alsocomprise the steps of:

-   -   iii) cooling the reaction solution to form a cooled solution of        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide;    -   iv) solidifying said        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide from        said cooled solution by adding a dialkyl ether; and    -   v) collecting said solid        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide.

However, it is the choice of the formulator which means is utilized toisolate the final product. For example, other additional steps which mayfacilitate improved yields and/or purity, include:

-   -   vi) treating the solid        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]-oxalamide        obtained in step (b)(vi) with hexane to form a slurry of        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]-oxalamide;        and    -   vii) collecting said        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]-oxalamide        from said slurry to form purified        N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]-oxalamide.

In another aspect, the inventions relate to a process for preparingN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide whichcomprises:

-   -   a) dissolving 2,4-dimethoxybenzylamine or an ammonium salt        thereof having the formula:

-   -   in triethylamine and toluene to form an admixture;    -   b) adding ethyl 2-chloro-2-oxoacetate having the formula:

to said admixture formed in step (a) at a temperature at or below about10° C., to form ethyl 2-(2,4-dimethoxybenzylamino)-2-oxoacetate havingthe formula:

and

-   -   c) reacting said ethyl 2-(2,4-dimethoxybenzylamino)-2-oxoacetate        formed in step (b) with 2-(pyridin-2-yl)ethylamine having the        formula:

to form a reaction solution and subsequently heating the reactionsolution to formN-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide; and

-   -   d) optionally, isolating the product of step (c) by:        -   i) cooling the refluxing solution to a temperature of from            about 25° C. to about 35° C. to form a cooled solution of            N-(2,4-dimethoxy-benzyl)-N′-[2-(pyridm-2-yl)ethyl]oxalamide;        -   ii) precipitating said            N-(2,4-dimemoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl3-oxalamide            from said cooled solution by adding methyl tert-butyl ether;            and        -   iii) collecting said            N-(2,4-dimethoxybenzyl)-N′-[2-(pyridin-2-yl)ethyl]oxalamide.

Other oxalamide compounds disclosed herein may be prepared by methodssimilar to those disclosed above.

Other improved methods of synthesis of synthesis disclosed herein relateto improved methods of preparing starting materials.

For example, some aspects of the inventions relate to improved processesof preparing 2-methoxy-4-methyl-benzylamine or a salt thereof, by thereaction sequence shown below:

comprising:

-   -   a) methylating 2-hydroxy-4-methyl-benzamide with a methylating        agent, to provide 2-methoxy-4-methyl-benzamide; and    -   b) reducing 2-methoxy-4-methyl-benzamide with a hydride reducing        agent, to provide 2-methoxy-4-methyl-benzylamine or a salt        thereof.s illustrated by the drawing below:

In such processes for producing precursors of oxalamide compounds, themethylating agent can include a variety of reagents well known to thoseof ordinary skill in the art, such as methyl halides, dimethyl sulfate,methyl tosylates, and the like. Similarly, a variety of hydride reducingagents (such as lithium aluminum hydride, lithium diisobutyl-aluminumhydride, lithium tri-tertbutoxide-aluminum hydride, or sodiumbis(2-methoxyethoxy)aluminum hydride, or similar boron hydride reagents)that will selectively reduce the carbonyl group of the amide to an amineare well known in the art. See for example Example 30-1.

Similarly, the inventions also relate to processes for producing otherprecursors for oxalamide compounds, as exemplified by a process ofpreparing 2-(5-methylpyridin-2-yl)ethamine or a salt thereof,comprising:

-   -   a) treating acetonitrile with a strong base to remove a hydrogen        ion therefrom,    -   b) condensing 2-bromo-5-methylpyridine with the base-treated        acetonitrile, to provide 2-(5-methylpyridin-2-yl)acetonitrile;        and    -   c) reducing the nitrile group of        2-(5-methylpyridin-2-yl)acetonitrile, to provide        2-(5-methylpyridin-2-yl)ethamine or a salt thereof. This process        is illustrated below:

In such processes, one of the hydrogens of acetonitvile can be removedwith very strong bases, such as alkyl or aryl lithium reagents, or alithium salt of a dialkyl amine, to generate highly nucleophilicorganometallic salts of acetonitrile, which can readily displace ahalogen from an aryl halide such as 2-bromo-5-methylpyridine, tosynthesize 2-(5-methylpyridin-2-yl)acetonitrile, which can be reduced tothe corresponding 2-(5-methylpyridin-2-yl)ethamine, by a variety ofknown stoichiometric hydride reducing agents, or by catalytichydrogenation. A well known catalyst for such catalytic hydrogenationsis Raney nickel.

Lastly, the final oxalamide compounds can be synthesized by sequentiallycondensing oxalamide precursors such as those exemplified above. Forexample, in a “one pot” procedure, such as that detailed in Example 30-1below.

Such a “one pot” process for preparingN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide,comprises:

-   -   a) reacting 2-methoxy-4-methylbenzylamine with        2-chloro-oxoacetate, to provide        N-(2-methoxy-4-methyl-benzyl)-oxalamic acid ethyl ester; and    -   b) reacting N-(2-methoxy-4-methyl-benzyl)-oxalamic acid ethyl        ester with 2-(5-methylpyridin-2-yl)ethamine, to provide        N¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide.

Both steps of “one pot” processes are typically conducted in a singlesolvent, such as acetonitrile, and a mild organic or inorganic base,such as triethylamine. However, Applicants have unexpectedly discoveredthat yields can sometimes be significantly improved by employing anisolation and purification procedure between the two steps, asillustrated by Example 30-2.

Lastly, Applicants have discovered that many of the amide and/oroxalamide compounds of the invention are largely insoluble in highlyapolar media such as hydrocarbons, fats, or oils, but can often bebeneficially recrystallized to high purity levels by dissolving andheating the compounds in a mixture of hepatane and ethyl acetate, thencooling the solution. In particular, as is described in detail inExample 30-2, the inventions relate to processes for crystallizingN-(heptan-4-yl)-benzo[d][1,3]dioxol-5-carboxamide, comprising:

-   -   c) dissolving a composition comprising        N-(heptan-4-yl)-benzo[d][1,3]dioxol-5-carboxamide in ethyl        acetate and heptane at elevated temperatures to form a solution;        and    -   d) cooling the solution, thereby forming a solid comprising        N-(heptan-4-yl)-benzo[d][1,3]dioxol-5-carboxamide.

In many embodiments of such purification processes, the crystallizationis conducted under an atmosphere of dry nitrogen, and the dissolvingstep carried out at a temperature of from about 40° C. to about 60° C.,then the resulting solution is cooled to a temperature from about 0° C.to about 30° C., then the resulting solid crystals are isolated anddried to yield a dry product that can be greater than 99% pureN-(heptan-4-yl)-benzo[d][1,3]dioxol-5-carboxamide.

Measuring the Biological Activity of the Compounds of the Invention

Cell based technologies and assays, such as those disclosed in WO02/064631 and WO 03/001876, and U.S. Patent Publication No. US2003/0232407 A1 were used both to initially screen a wide variety ofclasses of compounds for agonist or antagonist activity for T1R1/T1R3“savory” taste receptors, or T1R2/T1R3 “sweet” taste receptors that hadbeen expressed in appropriate cell lines. Once initial “hits” wereobtained for amide compounds in such cell lines, the same assays andalso certain cell and/or receptor-based assays were used as analyticaltools to measure the ability of the compounds of Formula (I) to enhancethe savory taste of MSG or the sweet taste of known sweeteners such assucrose, fructose, and were used to provide empirical data to guide aninterative process of synthesizing and testing structural variants ofthe amide compounds, in combination with occasional human taste testingof high interest compounds, so as to design, test, and identify speciesand genuses of compounds with increased and optimized levels ofdesirable biological activities.

Many embodiments of the inventions relate to the identification ofspecific compounds and classes of the amide compounds of Formula (I)that modulate (increase or decrease) the activity of the T1R1/T1R3(preferably hT1R1/hTIR3) savory taste receptor (umami receptor), aloneor in combination with another compound that activates hT1R1/hT1R3,e.g., MSG. Particularly, in many embodiments the invention relate to theamides of Formula (I) that modulate the activity of hT1R1/hT1R3 (humanumami receptor) in vitro and/or in vivo. In another aspect, theinvention relates to compounds that modulate the human perception ofsavory (umami) taste, alone or in combination with another compound orflavorant, when added to a comestible or medicinal product orcomposition.

In some embodiments of the invention, it has been very unexpectedlydiscovered that at least some of the amide compounds of Formula (I) canmodulate the human perception of umami taste, alone or in combinationwith another compound or flavorant composition, when added to acomestible or medicinal product or composition.

In Vitro Ht1R1/Ht1R3 Umami Taste Receptor Activation Assay

In order to identify new savory flavoring agents and enhancers,including compounds with savory agonist and enhancer activities (dualactivity), the compounds of Formula (I) were screened in primary assaysand secondary assays including compound dose response and enhancementassay. In a primary assay for potential ability to modulate umami taste,amide compounds of Formula (I) that can be either savory flavoringagents in their own right or flavor enhancers of MSG are identified andscores of their activities are given as percentage of the maximum MSGintensity (%). In compound dose response, an EC₅₀ is calculated toreflect the potency of the compound as a savory agonist or enhancer.

An HEK293 cell line derivative (See e.g., Chandrashekar, et al., Cell(2000) 100: 703-711) which stably expresses Gal5 and hT1R1/hTIR3 underan inducible promoter (see WO 03/001876 A2) was used to identifycompounds with savory tasting properties.

Compounds covered in this document were initially selected based ontheir activity on the hT1R1/hT1R3-HEK293-Gα15 cell line. Activity wasdetermined using an automated fluorometric imaging assay on a FLIPRinstrument (Fluorometric Intensity Plate Reader, Molecular Devices,Sunnyvale, Calif.) (designated FLIPR assay). Cells from one clone(designated clone 1-17) were seeded into 384-well plates (atapproximately 48,000 cells per well) in a medium containing Dulbecco'smodified Eagle's medium (DMEM) supplemented with GlutaMAX (Invitrogen,Carlsbad, Calif.), 10% dialyzed fetal bovine serum (Invitrogen,Carlsbad, Calif.), 100 Units/ml Penicillin G, 100 μg/ml Streptomycin(Invitrogen, Carlsbad, Calif.) and 60 μM mifepristone (to induceexpression of hT1R1/hT1R3, (see WO 03/001876 A2). 1-17 cells were grownfor 48 hours at 37° C. 1-17 cells were then loaded with the calcium dyeFluo-3AM (Molecular Probes, Eugene, Oreg.), 4 μM in a phosphate bufferedsaline (D-PBS) (Invitrogen, Carlsbad, Calif.), for 1.5 hours at roomtemperature. After replacement with 25 μl D-PBS, stimulation wasperformed in the FLIPR instrument and at room temperature by theaddition of 25 μl D-PBS supplemented with different stimuli atconcentrations corresponding to twice the desired final level. Receptoractivity was quantified by determining the maximal fluorescenceincreases (using a 480 nm excitation and 535 run emission) afternormalization to basal fluorescence intensity measured beforestimulation.

For dose-responses analysis, stimuli were presented in duplicates at 10different concentrations ranging from 1.5 nM to 30 μM. Activities werenormalized to the response obtained with 60 mM monosodium glutamate, aconcentration that elicits maximum receptor response. EC₅₀ ^(s)(concentration of compound that causes 50% activation of receptor) weredetermined using a non-linear regression algorithm, where the Hillslope, bottom asymptotes and top asymptotes were allow to vary.Identical results were obtained when analyzing the dose-response datausing commercially available software for nonlinear regression analysissuch as GraphPad PRISM (San Diego, Calif.).

In order to determine the dependency of hT1R1/hT1R3 for the cellresponse to different stimuli, selected compounds were subjected to asimilar analysis on 1-17 cells that had not been induced for receptorexpression with mifepristone (designated as un-induced 1-17 cells). Theun-induced 1-17 cells do not show any functional response in the FLIPRassay to monosodium glutamate or other savory-tasting substances.Compounds were presented to un-induced umami cells at 10 μM—or threetimes the maximum stimulation used in the dose-response analysis.Compounds covered in this document do not show any functional responsewhen using un-induced umami cells in the FLIPR assay.

In some aspects of the present invention, an EC₅₀ of lower than about 10mM is indicative of compounds that induce T1R1/T1R3 activity and isconsidered a savory agonist. Preferably a savory agonist will have EC₅₀values of less than about 1 mM; and more preferably will have EC50values of less than about 20 μM, 15 μM, 10 μM, 5 μM, 3 μM, 2 μM, 1 μM,0.8 μM or 0.5 μM.

In umami taste enhancement activity assay experiments, which produce an“EC₅₀ ratio” measurement of how effectively the amide compounds of theinvention enhance the savory flavorant (typically MSG) already in a testsolution. A series of measurements of the dose response is run insolutions comprising MSG alone, then a second dose response is run withMSG in combination with predetermined amounts of a candidate compound ofFormula (I) at the same time.

In this assay, increasing concentrations of monosodium glutamate(ranging from 12 μM to 81 mM) were presented, in duplicates, in thepresence or absence of a fixed concentration of the test compound.Typical compound concentrations tested were 30 μM, 10 μM, 3 μM, 1 μM,0.3 μM, 0.1 μM and 0.03 μM. The relative efficacy of compounds ofFormula (J) at enhancing the receptor was determined by calculating themagnitude of a shift in the EC₅₀ for monosodium glutamate. Enhancementwas defined as a ratio (EC₅₀R) corresponding to the EC₅₀ of monosodiumglutamate, determined in the absence of the test compound, divided bythe EC₅₀ of monosodium glutamate, determined in the presence of the testcompound. Compounds exhibiting EC₅₀R>2.0 were considered enhancers.

Stated alternatively, “EC₅₀ ratio” as compared to MSG is calculatedbased on the following definitions:

EC₅₀ Ratio vs. MSG=EC₅₀ (MSG)/EC₅₀ (MSG+[Compound]) wherein “[compound]”refers to the concentration of the compound of Formula (I) used toelicit (or enhance or potentiate) the MSG dose response.

It should be noted that the EC₅₀ ratio measured can depend somewhat onthe concentration of the compound itself. Preferred savory enhancerswould have a high EC₅₀ Ratio vs. MSG at a low concentration of thecompound used. Preferably the EC₅₀ ratio experiments to measure umamienhancement are run at a concentration of a compound of Formula (I)between about 10 μM to about 0.1 μM, or preferably at 1.0 μM or 3.0 μM.

An EC₅₀ ratio of greater than 1 is indicative of a compound thatmodulates (potentiates) hT1R1/hT1R3 activity and is a savory enhancer.More preferably, the savory taste enhancer compounds of Formula (I) willhave EC₅₀ ratio values of at least 1.2, 1.5, 2.0, 3.0, 4.0, 5.0, 8.0, or10.0, or even higher.

In one aspect, the extent of savory modulation of a particular compoundis assessed based on its effect on MSG activation of T1R1/T1R3 in vitro.It is anticipated that similar assays can be designed using othercompounds known to activate the T1R1/T1R3 receptor.

Specific compounds and generic classes of compounds that been shown tomodulate hT1R1/hT1R3 based on their EC50 ratios evaluated according tothe above formula are identified in the detailed description of theinvention, the examples, and the claims.

The procedures used for human taste testing of the umami/savorycompounds of Formula (I) are reported hereinbelow.

EXAMPLES

The following examples are given to illustrate a variety of exemplaryembodiments of the invention and are not intended to be limiting in anymanner.

For the purpose of this document, the compounds individually disclosedin the following Examples can be referred in shorthand by the number ofthe Example. For example, as shown immediately bellow, Example 1discloses a synthesis of a particular compound(N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide), and the results ofexperimental assays of its biological effectiveness, which compound isand can be referred to herein in shorthand form as Compound 1.

Example 1 N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide

To a solution of heptan-4-amine (8.06 mL, 54 mmol) in triethylamine(15.3 mL, 108 mmol) and dichloromethane (135 mL), was added, dropwise at0° C., a solution of benzo[1,3]dioxole-5-carbonyl chloride (10 g, 54mmol) dissolved in dichloromethane (135 mL). The reaction mixture wasstirred for 1 h. Solvent was removed under reduced pressure and theresidue was dissolved in EtOAc. The organic layer was washedsuccessively with 1 N aq. HCl, 1 N aq. NaOH, water, brine, dried (MgSO₄)and concentrated. The residue was recrystallized in EtOAc and Hexanes toafford 6.9 g of N-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide(48.3%) as a white solid. ¹H NMR (500 MHz, CDCl₃): δ 0.92 (t, 6H), 1.38(m, 6H), 1.53 (m, 2H), 4.11 (m, 1H), 5.63 (m, 1H), 6.01 (s, 2H), 7.98(d, 1H), 7.27 (s, d, 2H). MS (M+H, 264).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.2 μM, and when present at 0.03 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of6.92.

Example 1-1 Improved Preparation and Purification ofN-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide

In a clean fume hood, a 3-neck round bottom flask equipped with amechanical stirring assembly, addition funnel, thermocouple withdisplay, nitrogen inlet, and drying tube was placed in a cooling bath.The flask was also blanketed with a nitrogen atmosphere. To the flaskunder nitrogen, 674 g of 4-heptylamine (1 equiv., 5.85 moles) was added.THF (3.37 L) was then added to the flask and the reaction mixture wasstirred. Triethylamine (1184 g, 2 equiv., 11.7 moles) was next added tothe reaction mixture under nitrogen and the reaction mixture was cooledto an internal temperature of from minus 5° C. to 0° C.

In a polyethylene pail, piperonyl chloride (1080 g, 5.85 moles) wasdissolved in THF (3.37 L) and stored under a blanket of nitrogen. Thissolution was transferred to the addition funnel on the 3-neck flask. Thesolution was added to the reaction mixture in portions over 1-2 hours,keeping the internal temperature of the reaction mixture below 10° C.using external cooling as necessary. The piperonyl chloride in THFremained covered and blanketed with nitrogen between chargings. Afterthe addition was complete, the reaction mixture was heated to from 20°C. to 25° C. and stirred an additional 30 minutes. The reaction wasmonitored by HPLC. When the reaction was complete (<1.0% piperonylchloride remaining), methyl-t-butylether (6.73 L) was added with rapidstirring for 10 minutes. The resulting mixture was transferred to aseparatory funnel and washed with 1 N HCl (1.7 L). The organic layer waswashed with 1 N NaOH (1.7 L), water (3.37 L), and brine (1.7 L) anddried with 100 g of magnesium sulfate. The reaction mixture was filteredthrough a Buchner funnel and concentrated under vacuum with a bathtemperature of from 35° C. to 45° C. Heptane (1.76 L) was added to thecrude solid with stirring to form a thick slurry. The slurry wasfiltered through a Buchner and washed once with heptane (0.88 L). Thesolids were transferred to a clean dry drying tray and vacuum dried atfrom 40° C. to 45° C., until a constant weight was obtained.

A 3-neck round bottom flask equipped with a mechanical stirringassembly, condenser, thermocouple with display, nitrogen inlet, anddrying tube was placed in a heating mantle. The flask was blanketed witha nitrogen atmosphere and crudeN-(heptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide (1484 g) was addedunder nitrogen. Ethyl acetate (3.72 L) was also added. The reactionmixture was heated to an internal temperature—of from 50° C. to 55° C. Aclear solution was obtained. The solution was hot filtered through aBuchner funnel using a glass fiber filter on top of filter paper. A3-neck round bottom flask was equipped with a mechanical stirringassembly, addition funnel, thermocouple with display, nitrogen inlet,and drying tube and was placed in a heating mantle. The flask wasblanketed with a nitrogen atmosphere and the filtrate was transferred tothe flask and stirred. The reaction mixture was heated to an internaltemperature of from 40° C. to 50° C. Heptane (9.02 L Filtered through aBuchner funnel using a paper filter) was added in a steady stream over aminimum of 30 minutes while the internal temperature was maintained from40° C. to 50° C. After the addition was complete, the reaction mixturewas cooled to from 0° C. to 5° C. and kept at this temperature for 1hour. The solution was filtered through a Buchner funnel using apolypropylene filter pad and washed with cold heptane (1.13 L, Filteredthrough a Buchner funnel using a paper filter and cooled to 0° C.). Thesolids were transferred to a clean dry drying tray and vacuum dried atfrom 40° C. to 45° C. for a minimum of 14 hours, until a constant weightwas obtained. This protocol provided 1276 g of theN-(heptan-4-yl)ben2o[d][1,3]dioxole-5-carboxamide at greater than 99.9%purity (as determined by HPLC) for an overall yield of 86%.

Example 1-2

To a 500 mL 3-neck flask equipped with a magnetic stirrer, additionfunnel, thermocouple, drying tube, and a cooling bath under nitrogenblanketing was charged piperonylic acid (25 g, 150 mmol), CH₂Cl₂ (200mL) and DMF (2.5 mL). The resulting mixture was cooled to 0° C. andtbionyl chloride (18.8 g, 158 mmol) was added over approximately 10minutes. When the addition was complete the solution was heated toreflux for approximately 1 hour. The reaction was cooled to 0° C. andheld until used subsequently.

To a 1 L 3-neck flask equipped with a magnetic stirrer, addition funnel,thermocouple, drying tube, and a cooling bath under nitrogen blanketingwas charged 4-heptylamine (17.3 g, 150 mmol), triethylamine (30.5 g, 301mmol), CH₂Cl₂ (125 mL) and DMF (2.5 mL). The solution was cooled to 0°C. and the cold solution of acid chloride was added drop wise overapproximately 1 hour while maintaining the temperature below 10° C. Whenthe addition was complete, water (100 mL) was added while holding thetemperature below about 20° C. The contents of the reaction flask wasthen transferred to a reparatory funnel and the organic layer extractedwith 1N HCl (aq.) (2×40 mL), water (40 mL), 1N NaOH (aq.) (40 mL), water(40 mL), then NaCl (sat. aq.) (40 mL). The organic phase was dried overMgSO₄, filtered and concentrated to a reduced volume after which heptane(100 mL) was added and the solution concentrated to dryness underreduced pressure. The resulting crude product was treated with heptane(150 mL) and the resulting solid were collected by filtration, washedwith heptane (50 mL) then dried under vacuum to afford the desiredproduct. Yield was 37.4 g (94.4%).

Example 2 N-(2-raethylheptan-4-yl)benzo[d][1,3]dioxole-5-carboxamide

Prepared in a similar manner to example 1 usingbenzo[d][1,3]dioxole-5-carbonyl chloride and 2-methylheptan-4-amine(example 2a). ¹H NMR (500 MHz, CDCl₃): δ 0.93 (m, 9H); 1.38 (m, 5H);1.53 (m, 1H); 1.66 (m, 1H); 4.21 (m, 1H); 5.61 (d, 1H); 6.01 (s, 2H);6.82 (d, H); 7.26 (m, 2H). MS (278, M+H).

Preparation of 2-methylheptan-4-amine

To a solution of 2-methylheptan-4-one (4.24 g, 33.07 mmol), in methanol(60 mL), were added ammonium acetate (25.50 g, 330.71 mmol) and sodiumcyanoborohydride (2.08 g, 33.07 mmol). The reaction mixture was stirredat room temperature for about 24 hours. The solvent was removed underreduced pressure and the residue was diluted with water and basifiedwith 15% NaOH aqueous and extracted with ether. The extract was washedwith brine, dried over anhydrous magnesium sulfate, filtered andevaporated to give 3.3 g of 2-methylheptan-4-amine (77%). MS (M+H, 130).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.22 μM.

Example 3 N-(2-methylhexan-3-yl)benzo[d][1,3]dioxole-5-carboxamide

Prepared in a similar manner to example 1 usingbenzo[d][1,3]dioxole-5-carbonyl chloride and 2-methylhexan-3-amine(example 3a). ¹H NMR (500 MHz, CDCl₃): δ 0.93 (m, 9H); 1.37 (m, 3H);1.56 (m, 1H); 1.83 (m, 1H); 4.01 (m, 1H); 5.67 (d, 1H); 6.02 (s, 2H);6.82 (d, 1H); 7.28 (m, 2H). MS (M+H, 264).

a. 2-methylhexan-3-amine was prepared using the same procedure describedin example 2a starting from 2-methylhexan-3-one. Yield: 40%. ¹H NMR (500MHz, CDCl₃): δ 0.86 (d, 3H); 0.91 (m, 6H); 1.20-1.29 (m, 2H); 1.38-1.47(m, 2H); 1.47 (s, 2H); 1.58 (m, 1H); 2.51 (m, 1H). MS (M+H.116).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.61 μM.

Example 4 N-(2,3-dimethylcyclohexyl)benzo[d][l,31-dioxole-5-carboxamide

2,3-dimethylcyclohexanamine (20 μmol) andbenzo[d][1,3]dioxole-5-carboxylic acid (1.1 eq) were each dissolved inacetonitrile/dichloromethane (200 μL, 2:1). PS-Carbodiimide resin (2 eq)was loaded into a 1.2 mL 96 well Greiner plate, followed by the additionof amine and acid solutions. Hydroxybenzotriazole (1.1 eq) was dissolvedin DMF (100 mL) and was added into the reaction well. The reaction wasshaken overnight at room temperature. Once the reaction was completed,PS-Trisamine resin (1.5 eq) was added into the reaction mixture and thesolution was allowed to shake overnight at room temperature.Acetonitrile (200 mL) was added into the reaction well, and the topclear solution was transferred into a new plate. The solution wasevaporated to giveN-(2,3-dimethylcyclohexyl)benzo[d][1,3]dioxole-S-carboxamide. MS (M+H,276.20).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.45 μM, and when present at 1 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of8.4.

Example 5 (R)-methyl2-(benzo[d][1,3]dioxole-6-carboxamido)-4-methylpentanoate

Prepared in a similar manner to example 1 usingbenzo[d][1,3]dioxole-5-carbonyl chloride and D-leucine methyl esterhydrochloride. Yield: 83%. ¹H NMR (500 MHz, CDCl₃): δ 0.98 (m, 6H);1.63-1.67 (m, 1H); 1.71-1.76 (m, 2H); 3.76 (s, 3H); 4.83 (m, 1H); 6.03(s, 2H); 6.38 (d, 1H); 6.83 (d, 1H); 7.32 (s, 1H); 7.33 (d, 1H). MS(M+H, 294). m.p: 89-90° C.

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.34 μM, and when present at 0.1 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of4.9.

Example 6 (R)-methyl2-(benzo[d][1,3]dioxole-6-carboxamido)-3-methylbutanoate

Prepared in a similar manner to example 4 usingbenzo[d][1,3]dioxole-5-carboxylic acid and (R)-methyl2-amino-3-methylbutanoate. Yield: 50%. MS (M+H, 280.1).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 1.16 μM.

Example 7 N-(hexan-3-yl)-4-methoxy-3-methylbenzamide

Prepared in a similar manner to example 4 using4-methoxy-3-methylbenzoic acid and hexan-3-amine (example 28a). ¹H NMR(500 MHz, CDCl₃): δ 0.94 (m, 6H); 1.41 (m, 4H); 1.46 (m, 1H); 1.64 (m,1H); 2.24 (s, 3H); 3.87 (s, 3H); 4.08 (m, 1H); 5.69 (d, 1H); 6.83 (d,1H); 7.54 (s, 1H); 7.62 (d, 1H). MS (M+H, 250).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.12 μM.

Example 8 N-(heptan-4-yl)-6-methylbenzo[d][1,3]dioxole-5-carboxamide

Prepared in a similar manner to example 4 using6-methylbenzo[d][1,3]dioxole-5-carboxylic acid and heptan-4-amine. MS(M+H, 278.67).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.11 μM.

Example 9 N-(heptan-4-yl)-2-methylbenzo[d][1,3]dioxole-5-carboxamide

N-(heptan-4-yl)-3,4-dihydroxybenzamide (0.5 mmol) was dissolved intoluene (1.6 mL). P-Toluenesulfonic acid monohydrate (0.3 eq) was addedto the reaction, followed by addition of acetaldehyde (2 eq). Thereaction was performed using microwave (180 C, 300 W) and ran for 10minutes. The solvent was evaporated. The residue was dissolved inmethanol (1 mL) and purified by HPLC. Yield 20%, MS (M+H 278.10).

a. N-(heptan-4-yl)-3,4-dihydroxybenzamide was prepared in a similarmanner to example 4 using 3,4-dihydroxybenzoic acid and heptan-4-amine.Yield: 25%. MS (M+H, 252.1).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.1 μM, and when present at 0.03 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of3.68.

Example 10N-(heptan-4-yl)-2,2-dimethylbenzo[d][1,3]dioxole-5-carboxamide

Prepared in a similar manner to example 4 using sodium2,2-dimethylbenzo[d][1,3]dioxole-5-carboxylate and 4-heptylamine(example 10a). Yield 30%. ¹H NMR: 0.92 (t, 6H, J=7.2 Hz), 1.42 (m, 6H),1.53 (m, 2H), 1.68 (s, 6H), 4.12 (m, 1H), 5.61 (d, 1H, J=8.9 Hz), 6.72(d, 1H, J=8 Hz), 7.16 (d, 1H₅ J=1.5 Hz), 7.22 (dd, 1H, J=1.5 Hz, J=17Hz). MS (M+H, 292).

a. Sodium 2,2-dimethylbenzo[d][1,3]dioxole-5-carboxylate and4-heptylamine

Ethyl 2,2-dimethylbenzo[d][1,3]dioxole-5-carboxylate (example 10b) (461mg, 2.08 mmol) was stirred in dioxane (16 mL) and 1.0N aqueous NaOH(4.16 mL) for 20 hours at room temperature. The solvent was removedunder reduced pressure to afford the desired product (449 mg). (M−H,193).

b. Ethyl 2,2-dimethylbenzo[d][1,3]dioxole-5-carboxylate

Ethyl 3,4-dihydroxybenzoate (910.9 mg, 5 mmol) was combined with2,2-dimethoxypropane (1.23 mL, 10 mmol) and a catalytic amount ofp-toluene sulfonic acid in toluene. The mixture was heated to refluxusing a Dean-Stark trap for 20 hours. After solvent removal underreduced pressure, the crude was dissolved in ethyl acetate and washedsuccessively with a saturated aqueous solution of sodium bicarbonate,water, and brine. The organic layer was dried over anhydrous sodiumsulfate. Purification by chromatography on silica gel using a gradienthexane:ethyl acetate, 90:10 to 75:25, afforded a white powder (539.1 mg,49%). ¹H NMR (CDCl₃): 1.36 (t, 3H, J=7.2 Hz), 1.69 (s, 6H), 4.32 (q, 2H,J=7.1 Hz, J=14.2 Hz), 6.74 (d, 1H, d, J=8.2 Hz), 7.38 (d, Ih, J=1.7 Hz),7.61 (dd, 1H₅ J=1.8 Hz, J=8.3 Hz).

The compound had an ECso for activation of ahT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 2.7 μM.

Example 11 2,3-Dihydro-benzol[1,4]dioxine-6-carboxylic acid(1-propyl-butyL)-amide

Prepared in a similar manner to example 4 using2,3-Dihydro-benzo[1,4]dioxine-6-carboxylic acid and heptan-4-amine. MS(M+H, 278.2).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.49 μM.

Example 12 Preparation of (R)-methyl4-chloro-2-(5-methylbenzofuran-2-carboxamido)pentanoate

Prepared in a similar manner to example 4 using5-chlorobenzofuran-2-carboxylic acid and D-leucine methyl ester. MS(M+H, 324).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.82 μM.

Example 13 N-(heptan-4-yl)benzo[b]thiophene-2-carboxamide

Prepared in a similar manner to example 4 usingbenzo[b]thiophene-2-carboxylic acid and 4-hepthylamine. MS (M+H, 276).

The compound had an EC50 for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.21 μM.

Example 14 4-methyl-3-methylsulfanyl-N-(1-propylbutyl)benzamide

Prepared in a similar manner as example 4 using4-methyl-3-(methylthio)benzoic acid (example 14a) and 4-heptylamine.Yield: 50%. ¹HNMR (500 MHz, CDCl₃): δ 0.93 (t, 61-1, J=7.2 Hz),1.40-1.41 (m, 8H), 2.35 (s, 3H), 2.51 (s, 1H), 4.15 (m, 1H), 5.75 (d,1H, J=8.5 Hz), 7.15 (d, 1H, J=7.8 Hz), 7.31 (d, 1H, J=7.8 Hz), 7.65 (d,1H, J=1.5 Hz). MS (M+H, 280).

a. 4-methyl-3-(methylthio)benzoic acid: 3-Amino-4-methylbenzoic acid wassuspended in ice-water (55 mL), and concentrated HCl (8.56 mL) wasslowly added. An aqueous solution of sodium nitrite (2.4 g in 5.5 mL)was added to the suspension over a period of 15 minutes and the mixturewas stirred for another 15 minutes. Then, an aqueous solution of sodiumacetate (9.31 g in 18 mL) was added dropwise. The reaction was allowedto proceed for 45 min. A heavy orange precipitate was obtained. Theprecipitate was filtered off and washed with small portions of ice-coldwater. The solid was combined with a solution of potassium xanthogenate(11.93 g) and potassium carbonate (8.22 g) in 250 mL of water. Thereaction vessel was placed in a preheated oil bath at 70° C. and themixture was stirred for 25 minutes. The reddish solution was taken outof the bath and stirred for 15 minutes or until the temperature reached30° C. Sodium hydroxide (0.782 g) was added and stirred to dissolution.Dimethylsulfate (5.70 mL) was added. The mixture was stirred for 1 hourat room temperature then briefly refluxed. Solvent removal under reducedpressure yielded an orange solid. The solid was treated with a 2.0 Nsolution of H₂SO₄ and extracted with EtOAc. The extracts were washedwith water then dried over anhydrous MgSO₄. The solvent was removedunder reduced pressure to give a reddish crude solid. The solid wasadsorbed on silica gel and purified by column chromatography (gradient 5to 50% ethyl acetate in hexane) to give 4-methyl-3-(methylthio)benzoicacid as a pale yellow powder (2 g). ¹H NMR (500 MHz, CDCl₃): δ 2.39 (s,3H), 2.54 (s, 3H), 7.24 (d, 1H, J=7.8 Hz), 7.79 (d, 1H, J=7.8 Hz), 7.86(d, 1H, J=1.5 Hz).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.21 μM.

Example 15 4-methoxy-3-methyl-N-(2-methylheptan-4-yl)benzamide

Prepared in a similar manner as described in example 4 using4-methoxy-3-methylbenzoic acid and 2-methyl-4-heptanamine (example 2a).Yield: 45%. ¹H NMR (500 MHz, CDCl₃): δ 0.93 (m, 9H); 1.39 (m, 5H); 1.53(m, 1H); 1.67 (m, 1H); 2.24 (s, 3H); 3.86 (s, 3H); 4.23 (m, 1H); 5.64(d, 1H); 6.82 (d, 1H); 7.54 (s, 1H); 7.61 (d, 1H). MS (278, M+H).

The compound had an EC50 for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.1 μM.

Example 16 (R)-methyl 2-(3-chloro-4-methoxybenzamido)-4-methylpentanoate

Prepared in a similar manner to example 4 using 3-chloro-4-methoxybenzoic acid and D-leucine methyl ester hydrochloride. MS (M+H, 314.10).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.08 and when present at 0.01 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of13.18.

Example 17 3,4-Dimethoxy-N-(1-propyl-butyl)-benzamide

Prepared in a similar manner to example 4 using 3,4-dimethoxy benzoicacid and heptan-4-amine. MS (M+H, 279.37).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.36 μM.

Example 18 (R)—N-(1-methoxy-4-methylpentan-2-yl)-3,4-dimethylbenzamide

To a solution of(R)—N-(1-hydroxy-4-methylpentan-2-yl)-3,4-dimethylbenzamide (1.59 g,6.39 mmol) (example 18a) in dry DMF (20 mL) was added powdered NaOH (281mg, 7 mmol) an the solution was stirred at 0° C. for 2 hrs. Iodomethane(1 eq, 6.39 mmol) was added in DMF (10 ml) drop-wise over period of 1hr. The temperature was kept at 0° C. and the mixture was stirred for 1hr. The reaction was quenched by adding 300 ml of water. The aqueouslayer was extracted with dichloromethane, dried over MgSO₄ andevaporated. The residue was purified by flash chromatography onsilica-gel (toluene-ethyl acetate; 5-20% gradient) to give 1.23 g(R)—N-(1-methoxy-4-methylpentan-2-yl)-3,4-dimethylbenzamide (73%). ¹HNMR (500 MHz, CDCl₃): ÿ 0.94-0.97 (t, 6H), 1.41-1.47 (M, 1H), 1.54-1.60(m, 1H), 1.64-1.68 (m, 1H), 2.29 (d, 6H)₅ 3.36 (s₃ 3H), 3.45-3.50 (m,2H), 4.34-4.39 (m, 1H), 6.23-6.25 (d, 1H), 7.16-7.17 (d, 1H), 7.47-7.49(dd, 1H), 7.56 (s, 1H). MS (M+H, 264.3).

a. (R)—N-(1-hydroxy-4-methylpentan-2-yl)-3,4-dimethylbenzajnide wasprepared in a similar manner as described in example 4 using3,4-dimethylbenzoic acid and with (R)-aminoleucinol. Yield: 75%. MS(M+H, 250.3).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.2 μM.

Example 19 (R)-methyl2-(2,3-dimethylfuran-5-carboxamido)-4-methylpentanoate

Prepared in a similar manner to example 4 using4,5-dimethyl-furan-2-carboxylic acid and D-leucine methyl ester. Yield:27%. ¹H NMR (500 MHz, CDCl₃): ÿ 0.96 (t, 6H), 1.66 (m, 3H), 1.96 (s,3H), 2.26 (s, 3H), 3.75 (s, 3H), 4.78 (m, 1H), 6.51 (d, 1H), 6.89 (s,1H). MS (M+H, 268).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.59 μM.

Example 20 (R)-methyl2-(2,6-dimethoxyisonicotinamido)-4-methylpentanoate

Prepared in a similar manner to example 4 using2,6-Dimethoxy-isonicotinic acid and D-leucine methyl ester. ¹H NMR (500MHz, CDCl₃): δ 0.92 (d, 3H, J=7.27 Hz), 0.93 (d, 3H, J=7.26 Hz),1.41-1.58 (m, 8H), 3.95 (s, 3H), 4.08 (s, 3H), 4.15 (m, 1H), 6.43 (d,1H, J=8.32 Hz), 7.47 (m, broad, 1H), 8.41 (d, 1H, J=8.34 Hz). MS (M+H;311).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 1.91 μM.

Example 21 (S)—N-(2,3-Dihydro-1H-inden-1-yl)-4-methoxy-3-methylbenzamide

Prepared in a similar manner to example 4 using4-methoxy-3-methylbenzoic acid and (S)-2,3-dihydro-1H-inden-1-amine.Yield 63%. ¹HNMR (500 MHz, dMSO): δ 1.94-1.99 (m, 1H), 2.17 (s, 3H),2.41-2.46 (m, 1H), 2.82-2.87 (m, 1H), 2.96-3.01 (m, 1H), 3.83 (s, 3H),5.53-5.57 (dd, 1H), 6.98-6.99 (d, 1H), 7.16-7.23 (m, 3H), 7.26-7.27 (m,1H), 7.75-7.80 (m, 2H), 8.54-8.55 (d, 1H). MS (M+H, 282).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.08 μM.

Example 22(R/S)-4-Methoxy-N-(5-methoxy-2,3-dihydro-1H-inden-1-yl)-3-methylbenzamide

Prepared in a similar manner to example 4 using4-methoxy-3-methylbenzoic acid and5-methoxy-2,3-dihydro-1H-inden-1-amine (Example 121-2a) (47%). MS(M+H_(s) 312).

The compound had EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.08 μM.

Example 22-2a 5-methoxy-2,3-dihydro-1H-inden-1-amine

5-Methoxy-2,3-dihydromden-1-one (Ig, 6.17 mmol) was added to a solutionof hydroxylamine HCl (730 mg, 10.5 mmol) in 10 ml of water. The mixturewas brought up to 70° C. and a solution of sodium acetate (1.4 g, 16.7mmol) in 7 mL of H₂O, 14 ml of MeOH, 3 ml of THF was added. Afterstirring for 1.5 h at 70° C., 10 ml of H₂O was added to produce aprecipitate and the suspension was allowed to stir for 2 h. Theprecipitate was collected by filtration to give5-methoxy-2,3-dihydroinden-1-one oxime almost quantitatively and wasused in the next step without further purification. The oxime (0.5 g,2.82 mmol) was dissolved in MeOH and a catalytic amount of Raney nickeland 25 mL of ammonia solution in MeOH (7N) was added. The reaction wasstirred at r.t. overnight under H₂. The slurry was filtered over celiteand concentrated in vacuo, diluted with EtOAc, washed with water andbrine, dried over MgSO₄, filtered, and concentrated in vacuo to give thecrude title amine (yield, 45%). The crude amine was used without furtherpurification.

Numerous amide compounds of Formula (I) that fall within the subgenus of“oxalamide” compounds described elsewhere herein were also synthesizedand experimentally tested for effectiveness as activator of ahT1R1/hT1R3 umami receptor expressed in an HEK293 cell line.

Example 23 General procedure A for the preparation of an oxalamideSynthesis of N-(2-Methoxy-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide

2-Methoxybenzyl amine (5 mmol) was mixed with triethylamine (2 equiv.)in anhydrous Dioxane. Ethyl oxalyl chloride (1 equiv.) was added and themixture was shaken at room temperature for 0.5-2 hours. Then2-(2-pyridinyl)ethyl amine (1 equiv.) was added and the suspension washeated at 80° C. overnight. The solution was concentrated and theresidue was dissolved in ethyl acetate and washed with water. Theorganic layer was dried by sodium sulfate and solvent was evaporated togive the crude product, which was purified by flash columnchromatography to afford the title compound: yield 70%, m.p. 118-119°C.; m/e=314 [M+1]; 1H NMR (CDCl₃): 3.02 (t, 2H), 3.76 (dt, 2H), 3.86 (s,3H), 4.47 (d, 2H), 6.80-6.90 (m, 2H), 7.14-7.18 (m, 2H), 7.20-7.30 (m,2H), 7.55-7.62 (m, 1H), 7.75-7.83 (m, 1H), 8.05-8.12 (m, 1H), 8.55-8.63(m, 1H).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.34 μM, and when present at 0.3 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of18.85.

Example 24 N-(2,4-Dimethoxy-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide

Prepared in a similar manner to example 23 using 2,4-dimethoxybenzylamine, ethyl oxalyl chloride and 2-(2-pyridinyl)ethyl amine. Yield 72%,m.p. 123-124° C.; m/e=344 [M+1]; ¹H NMR (CDCl₃): δ 3.02 (t, 2H); 3.73(dd, 2H); 3.78 (s, 3H); 3.82 (s, 3H); 4.38 (d, 2H) 6.40 (dd, 1H); 6.44(d, 1H); 7.14 (m, 3H); 7.59 (m, 1H); 7.82 (t, 1H); 8.11 (t, 1H); 8.56(d, 1H); ¹³C NMR: δ 36.9, 38.9, 39.4, 55.6, 55.6, 98.8, 104.1, 117.8,121.9, 123.5, 130.7, 136.8, 149.6, 158.8, 158.8, 159.6, 160.1, 161.0.

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.09 μM, and when present at 0.3 μMenhanced the effectiveness of monosodium glutamate with an EC₅₀ ratio of6.51.

Example 24-1 Improved Preparation and Purification ofN-(2,4-Dimethoxy-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide

A clean and dry jacketed reactor was equipped with a condenser, aClaisen adapter, a temperature probe, and an addition funnel or headcolumn. The reactor was flushed with nitrogen gas for at least 15minutes. The reactor was then charged with 1,396 g (1 equiv., 8.3 mol)of 2,4-dimethoxybenzylamine, 1,693 g (2 equiv., 16.7 mol) oftriethylamine, and 25,086 mL of THF. The mixture was cooled to from 0 to5° C. Ethyl chlorooxoacetate (1140 g, 1 equiv., 8.3 mol) was added to anaddition funnel or head column and charged to the batch at such a ratethat the internal temperature did not exceed 10° C. Solids formedapproximately ⅓ into the addition. After the addition was complete, thecooling was turned off and the slurry was allowed to stir for 30 minutesat from 5° C. to 15° C. When complete, the reaction was warmed to from20° C. to 25° C. The organic phase was washed twice with 22,580 mL of 1N HCl. The organic phase was then washed with 12,600 mL saturated sodiumbicarbonate. Next, the organic phase was washed with 12,463 mL of brineand dried over magnesium sulfate. The mixture was filtered through athin pad of celite and concentrated at from 40° C. to 45° C. to a yellowoil. A total of 2,052 g (7.7 mol, 92%) of the crudeN-(2,4-dimethoxy-benzyl)-oxalamic acid ethyl ester was isolated.

In a clean fume hood, a 3-neck round bottom flask equipped with amechanical stirring assembly, addition funnel, thermocouple withdisplay, nitrogen inlet and drying tube was placed in a heating mantle.The flask was blanketed with a nitrogen atmosphere.N-(2,4-Dimethoxy-benzyl)-oxalamic acid ethyl ester (1116 g, 1 equiv.,4.18 moles), acetonitrile (12.5 L), and 2-(2-pyridyl)-ethylamine (1equiv., 4.18 moles) were added to the flask under nitrogen. The solutionwas heated to reflux and this temperature was maintained for a minimumof 20 hours (81° C.). After the reaction was complete, the reactionmixture was transferred to a cold water bath and cooled to 65° C. Thesolution was next transferred to a round bottom flask and concentratedunder vacuum with a bath temperature of from 40° C. to 45° C. Theresidue was dissolved in 1N HCl (16 L×2 separatory funnels), transferredto a separatory funnel and extracted with isopropyl acetate. (13.95 L×2separatory funnels). The combined organic layers were extracted with 1NHCl 13.61 L×2 separatory funnels). The combined aqueous layers werewashed with isopropyl acetate (3.4 L×2 separatory funnels) andtransferred to a 3-neck round bottom flask placed in a cooling bath witha mechanical stirring assembly, addition funnel, and thermocouple withdisplay. 6N NaOH was added to the flask via the addition funnel, and thereaction mixture was maintained at from 20° C. and 30° C. Methylenechloride was added to dissolve the resulting slurry (9.82 L) and theresulting clear solution was transferred to a separatory funnel. Theorganic phase was collected and washed with aqueous NaOH (6.88 L), driedover K₂CO₃, and filtered through a Buchner funnel using a polypropylenecloth filter. The solution was transferred to a round bottom flask andconcentrated under vacuum with a bath temperature of from 35° C. to 40°C. The solids were transferred to a clean dry drying tray and vacuumdried at from 40° C. to 45° C. until a constant weight was obtained.

A 3-neck round bottom flask equipped with a mechanical stirringassembly, condenser, thermocouple with display, nitrogen inlet, anddrying tube was placed in a heating mantle. The flask was blanketed witha nitrogen atmosphere. 1317 g of crudeN-(2,4-Dimethoxy-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide was addedunder nitrogen and then 18.7 L of ethyl acetate was added. The reactionmixture was heated to an internal temperature of from 50° C. and 55° C.until a clear solution was obtained. The heat was removed and heptanewas added via the addition funnel in a steady stream. After addition wascomplete, the reaction was placed in an ice bath and cooled to from 20°C. to 25° C. and maintained at this temperature for a minimum of 30minutes. The material was filtered through a Buchner funnel using apolypropylene filter and transferred to a 2:3 mixture of heptane inethyl acetate (7 L). The slurry was stirred for 30 minutes and filteredthrough a Buchner funnel using a polypropylene filter. Solids wererinsed with 800 mL of the 2:3 mixture of heptane in ethyl acetate anddried. The recrystallization wash procedure was repeated to afford 1079g of the final product (63% yield) in greater than 99.5% purity asdetermined by HPLC. An additional recrystallization was occasionallynecessary to obtain purities greater than or equal to 99.5%. In thesecases the material was recrystallized from 8:2 ethyl acetate to heptanein a manner similar to that described above to produce the product ingreater than 99.5% purity.

This recrystallization method can be used for the purification of otheroxalamide analogs disclosed herein.

Example 24-2 Improved Preparation and Purification ofN-(2,4-Dimethoxy-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide

To a 12 L 3-neck round bottom flask equipped with a mechanical stirrer,thermocouple, addition funnel, drying tube, and nitrogen blanketing,immersed in a cooling bath, was charged 2,4-dimethoxybenzylaminehydrochloride (500 g, 2.45 mol) and toluene (6 L). Triethylamine (1027mL, 7.36 mol) was slowly added and the solution stirred for 1 hour atambient temperature after which the reaction vessel was placed in amethanol/ice bath and the solution cooled to 0° C. Ethyl2-chloro-2-oxoacetate (273 mL, 2.45 mol) was added over approximately 25minutes while maintaining the reaction temperature below about 10° C.The reaction solution was then warmed to 22° C. and stirred at ambienttemperature overnight. [The course of the reaction was monitored by gaschromatography by collecting an aliquout of the reaction solution andpassing it through a filter syringe then subsequently rinsing the saltswith a 1:1 mixture of TEA/THF. Reaction was judged complete when lessthan 1% of the 2,4-dimethoxybenzylamine is present]

Once the reaction was judged complete, the solution was re-cooled to 15°C. and 1N HCl (aqueous) (3 L) was added at a rate which did not allowthe temperature to rise above about 23° C. The reaction solution wastransferred to a polyethylene crock and slurried in toluene (2.5 to 4mL/g of starting materials) and collected by filtration and the filtratewas set aside. The solids are re-slurried in toluene (2.5 to 4 mL/g ofstarting materials) and the liquid filtrate again collected byfiltration.

The combined filtrates were transferred to a separatory funnel and theorganic phase decanted. The solids were treated again with toluene andthe organic layer decanted. The combined organic layers were washed with1N HCl (aq.) (2×2 L), NaHCO₃ (sat. aq.) (2×2 L), NaCl (sat. aq.) (2 L),dried over MgSO₄, and filtered. The filtrate was charged to a 12 L3-neck round bottom flask equipped with a mechanical stirrer,thermocouple, addition funnel, drying tube, and heating mantle undernitrogen blanketing. 2-(2-Aminoethyl)pyridine (292 mL) was slowly addedmaintaining the temperature between 15° C. and 25° C. After addition wascomplete the reaction mixture was heated to reflux overnight. Thereaction vessel was then cooled to approximately 35° C. and methyltert-butyl ether (6.3 L) was added and the mixture cooled toapproximately 20° C. after which the product began to precipitate. Thesolution was held at 10° C. for 4 hours then at 0° C. to 5° C. for anadditional 30 minutes. The product was collected by filtration andwashed with heptane (4×6 L) to afford 610 g of crude product. The crudematerial was charged to a 12 L 3-neck round bottom flask equipped with amechanical stirrer, thermocouple, addition funnel, drying tube, andheating mantle under nitrogen blanketing. Ethyl acetate (9.15 L, 15 mL/gof crude material) was added and the solution was heated until a clearsolution was obtained. Additional heptane (6.71 mL) was added and theresulting slurry was cooled to about 20° C. and stirred for 30 minutes.The resulting solid was collected by filtration then re-slurried in amixture of heptane/ethyl acetate (2:3) (6.1 L). The solids were againcollected by filtration, the filter cake washed with heptane/ethylacetate (370 mL) and the collected product dried to a constant weight.Yield was 356 g (42%).

Example 25 General Procedure B for the Synthesis of an OxalamideN-(4-methyl-benzyl)-N′-(2-pyridin-2-yl-ethyl)-oxalamide

4-Methylbenzyl amine (1 mmol) was allowed to react with ethyl oxalylchloride (1 equiv.) in the presence of triethyl amine (2 equiv.) inacetonitrile at room temperature for 0.5-1 hour. Then2-(2-pyridinyl)ethyl amine (1 equiv.) was added and the suspension washeated at 160° C. in a microwave reactor for 5 minutes. The reactionmixture was subject to preparative HPLC to give the pure titleoxalamide: yield 60%; m.p. 152-154° C.; m/e=298 [M+1]; ¹H NMR (CDCl₃): δ2.33 (s, 3H), 3.10 (t, 2H), 3.75 (dt, 2H), 4.43 (d, 2H), 7.10-7015 (m,4H), 7.18-7.22 (m, 2H), 7.65-7.73 (ra, 2H), 8.12 (b, 1H), 8.60 (d, 1H).

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.41 μM.

Example 26N-(2-methoxy-4-methylbenzyl)-N′-(2-(pyridin-2-yl)ethyl)oxalamide

Prepared in a similar manner to example 25 using(2-methoxy-4-methylphenyl)methanamine (example 132a), ethyl oxalylchloride, and 2-(2-pyridinyl)ethyl amine, yield 20%. m.p: 128-131° C.;m/e=328 [M+1]; ¹H NMR (CDCl₃): 2.33 (s, 3H); 3.02 (t, 2H); 3.73 (m, 2H);3.84 (s, 3H); 4.42 (d, 2H); 6.70 (m, 2H); 7.14 (m, 3H); 7.60 (m, 1H);736 (s, 1H); 8.09 (s, 1H); 8.56 (d, 1H).

a. (2-methoxy-4-methylphenyl)methanamine: To a solution of2-methoxy-4-methylbenzamide (example 132b) (200 mg, 1.21 mmol) in THF(0.5 mL) was added 1 M BH₃.THF (2.4 ml, 2.42 mmol) slowly at roomtemperature. The resulting mixture was heated in a microwave reactor at130° C. for 7 min. Then 6 N aqueous HCl (1 mL) was added dropwise atroom temperature. The resulting mixture was heated in a microwavereactor at 120° C. for 4 min. The reaction mixture was washed with Et₂O(3×3 mL), then cooled to 0° C. and 10 N aqueous NaOH (0.8 mL) was added.The aqueous solution was saturated with K₂CO₃. The product was extractedwith CHCl₃ (6×5 mL). The organic extracts were dried (1:1 K₂CO₃/Na₂SO₄),filtered, concentrated in vacuo to afford 180 mg of(2-methoxy-4-methylphenyl)methanamine which was used directly.

b. 2-methoxy-4-methylbenzamide: 2-methoxy-4-methylbenzoic acid (500 mg,3.01 mmol) was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (577 mg, 3.01 mmol) and 1-hydroxybenzotriazole (407 mg,3.01 mmol) in 25 ml of dichloromethane at r.t. and stirred for 5 min. 2Mammonia solution in methanol (4.5 ml, 9.03 mmol) was added, the reactionmixture was stirred at r.t. for about 5 hr. then it was diluted withdichloromethane, washed with 1N HCl, sat. NaHCO₃, water and brine, driedover MgSO₄, filtered and evaporated to give 440 mg of2-methoxy-4-methylbenzamide, yield 88%.

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.04 uM.

Example 27 N-(2,4-dimethylbenzyl)-N′-(2-(pyridin-2-yl) ethyl)oxalamide

Prepared in a similar manner to example 25 using(2,4-dimethylphenyl)methanamine (example 133a), ethyl oxalyl chloride,and 2-(2-pyridinyl)ethyl amine, yield 60%; m.p. 148-149° C.; m/e=312[M+1]; ¹H NMR (CDCl₃): 2.28 (s, 3H); 2.30 (s, 3H); 3.05 (t, 2H); 3.76(dd, 2H); 4.43 (d, 2H); 6.99 (m, 2H); 7.11 (d, 1H); 7.17 (m, 2H); 7.54(s, 1H); 7.62 (m, 1H); 8.17 (s, 1H); 8.58 (d, 1H).

a. (2,4-Dimethylphenyl)methanamine: Lithium aluminum hydride IM solutionin THF (15.2 ml, 15.2 mmol) was placed in a pre-dried flask under argonat 0° C.; a solution of 2,4-dimethylbenzonitrile (1.0 g, 7.6 mmol) in 15ml of anhydrous ether was added drop wisely. After the addition, thereaction mixture was warmed up slowly to r.t. and stirred for 3 hr. thenit was cooled to 0° C., anhydrous sodium sulfate was added, and 1 ml ofwater was added drop wisely. The mixture was diluted with ethyl acetate,the insoluble matter was filtered out, the filtrate was washed withwater and brine, dried over MgSO₄, filtered and evaporated to give 1.03g of pure (2,4-dimethylphenyl)methanamine in quantitative yield withoutpurification.

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.07 μM.

Example 28N-(4-ethoxy-2-methoxybenzyl)-N′-(2-(pyridin-2-yl)ethyl)oxalamide

Prepared in a similar manner to example 25 using(4-ethoxy-2-methoxyphenyl)methanamine (example 134a), ethyl oxalylchloride, and 2-(2-pyridinyl)ethyl amine; yield 10%; m.p. 117-118° C.;m/e=358 [M+1]; ¹H NMR (CDCl₃): 1.40 (t, 3H); 3.03 (t, 2H); 3.74 (dd,2H); 3.82 (s, 3H); 4.01 (dd, 2H); 4.39 (d, 2H); 6.39 (d, 1H); 6.44 (s,1H); 7.15 (m, 3H), 7.61 (m, 1H); 7.81 (s, 1H); 8.10 (s, 1H); 8.56 (d,1H).

a. (4-ethoxy-2-methoxyphenyl)methanamine: To a solution of4-ethoxy-2-methoxybenzaldehyde (example 134b) (880 mg, 4.88 mmol) in 50ml of anhydrous methanol, were added ammonium acetate (7.5 g, 97.60mmol) and sodium cyanoborohydride (613 mg, 9.76 mmol). The reactionmixture was stirred at r.t. for about 4 hr. then it was concentrated ona rotary evaporator, the residue was diluted with water and basifiedwith 15% aqueous NaOH, extracted with ethyl acetate, washed with waterand brine, dried over MgSO₄, filtered and the solvent was evaporated,the residue was column chromatographed on silica gel (DCM/MeOH 9:1) toafford 150 mg of product; yield 17% (The method was not optimized).

b. 4-Ethoxy-2-methoxybenzaldehyde: To a solution of4-hydroxy-2-methoxybenzaldehyde (1.0 g, 6.57 mmol) in 10 ml of acetone,was added potassium carbonate (0.91 g, 6.57 mmol) and iodoethane (1.6ml, 19.71 mmol), the reaction mixture was stirred at r.t. over night.Acetone was removed on a rotary evaporator; the residue was diluted withwater and ethyl acetate; extracted with ethyl acetate, washed withbrine, dried over MgSO₄, filtered and evaporated to give crude product,which was column chromatographed on silica gel (ethylacetate/hexane=1:4) to give 943 mg of product; yield 80%.

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.1 μM.

Example 29 N-(2-chlorobenzyl)-N′-(2-(pyridin-2-yl)ethyl)oxalamide

Prepared in a similar manner to example 25 using(2-chlorophenyl)methanamine, ethyl oxalyl chloride, and2-(2-pyridinyl)ethyl amine; yield 45%; m/e=318 [M+1].

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.01 μM.

Example 30N¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide

¹H NMR (CDCl₃, 500 MHz): δ 2.29 (3H, s); 2.33 (3H, s); 2.97 (2H, t,J=6.5 Hz); 3.71 (2H, q, J=6.5 Hz); 3.83 (3H₅ s); 4.40 (2H, d, J=6.2 Hz);6.68 (1H, s); 6.69 (1H, d, J=7.7 Hz); 7.02 (1H, d, J=7.9 Hz); 7.09 (1H,d, J=7.5 Hz); 7.40 (1H, dd, J₁=1.8 Hz, J₂=7.8 Hz); 7.85 (1H, br t); 8.06(1H, br t); 8.38 (1H, s, J=7.5 Hz).

13C NMR (CDCl₃, 500 MHz): 18.3, 21.8, 36.5, 39.1, 39.6, 55.5, 111.5,121.3, 122.3, 123.0, 129.9, 131.3, 137.4, 139.6, 150.0, 155.7, 157.7,159.7, 160.1.

Elemental Analysis: Calculated for Ci₈H₂₁N₃O₃.¼H₂O: C, 65.97; H, 6.85;N, 12.15. Found: C, 66.10; H, 7.34; N, 12.17. MS (342, M+1). Whitepowder, melting point=133.5-134° C.

The compound had an EC₅₀ for activation of a hT1R1/hT1R3 umami receptorexpressed in an HEK293 cell line of 0.03 μM.

The compound was synthesized via the reaction sequence illustrated inthe diagram below, and the details of each of the six synthetic stepsare subsequently provided below.

Step 1: To a solution of 2-hydroxy-4-methylbenzoic acid (25 g, 0.164mol) in acetone (350 mL) was added K₂CO₃ (68 g, 0.492 mmol) followed byMeI (41 mL, 0.656 mmol) and the reaction mixture heated at reflux for 48hrs. After cooling to r.t. the reaction mixture was filtered and thefiltrate was evaporated to give the crude methyl2-methoxy-4-methylbenzoate. KOH (11.3 g, 1.2 eq) was dissolved in MeOH(300 mL) and the crude ester was added to the mixture and the solutionheated at reflux 48 hrs. After cooling the reaction mixture wasacidified with aq. HCl (1N) and extracted with ethyl acetate. Theorganic layer was washed with brine, dried over MgSO₄, filtered andevaporated. The residue was triturated with Ethyl acetate/Hexane to give20 g of 2-methoxy-4-methylbenzoic acid as a cream white solid (85%yield)

Step 2: To a mixture of 2-methoxy-4-methylbenzoic acid (20 g, 120.4mmol), EDC (23.1 g, 120.4 mmol) and HOBt (16.3 g, 120.4 mmol) indichloromethane (1 L) was added NH₃ (7N in MeOH, 52 mL, 3 eq) dropwise.The reaction mixture was stirred at room temperature overnight thenwashed successively with HCl (1N), saturated aq.NaHCO₃, water and brine,dried over MgSO₄, filtered and evaporated. The residue wasrecrystallized from ethyl acetate/hexane to give 16.5 gr of2-methoxy-4-methylbenzamide (83% yield).

Step 3: To a solution of 2-methoxy-4-methylbenzamide (14.55 g, 88.08mmol) in dry THF (50 mL) was added dropwise Borane-tetrahydrofurancomplex (1.0 M in THF, 220 mL, 2.5 eq) at 0° C. under N₂ atmosphere. Thereaction mixture was then heated to 60° C. overnight. The reaction wascooled to room temperature, aq.HCl (6 N, 37 mL) was added carefully andthe reaction mixture was then heated at 70° C. for 2 hrs. After cooling,water was added and the resulting solution was washed with ether. Theaqueous layer was basified with aq. NaOH (10 N) at 0° C. and saturatedwith K₂CO₃ then extracted with ethyl acetate. The organic layer waswashed with brine, dried over MgSO₄, filtered and evaporated to give 8.5g of (2-methoxy-4-methylphenyl)methanamine. (64% yield)

Step 4: To a solution of anhydrous acetonitrile (10.1 mL, 191.83 mmol,3.3 eq) in dry THF (500 mL) was added dropwise n-BuLi (2.5 M in Hexane,69.8 mL, 174.39 mmol, 3 eq) at −78° C. under N₂ atmosphere. Theresulting white suspension was stirred at −78° C. for 1 hr, and then asolution of 2-bromo-5-methylpyridine (10.0 g, 58.13 mmol, 1 eq) in dryTHF (30 mL) was added. The reaction mixture was kept at −78° C. for 1 hrthen warmed up slowly to r.t and stirred for another 1 hr. Ice/water wasadded and the layer was separated. The organic layer was washed withwater and brine, dried over MgSO₄, filtered and evaporated to give 18 gof crude 2-(5-methylpyridin-2-yl)acetonitrile. Since the product is veryvolatile, it was not dried under high vacuum and still contains somesolvent.

Step 5: To a solution of 18 g of crude2-(5-methylpyridin-2-yl)acetonitrile in dry THF (100 mL) was addeddropwise Borane-tetrahydrofuran complex (1.0 M in THF, 232 mL, 232.5mmol, 4 eq) at 0° C. under N₂ atmosphere. The reaction mixture was thenheated to 60° C. overnight. The reaction was cooled to room temperature,aq.HCl (6 N, 40 mL) was added carefully and the reaction mixture wasthen heated at 70° C. for 2 hrs. After cooling, water was added and theresulting solution was washed with ether. The aqueous layer was basifiedwith aq. NaOH (10 N) at 0° C. and saturated with K₂CO₃ then extractedwith ether (5×100 mL). The organic layer was dried over MgSO₄, filteredand evaporated to give 7.6 g of crude2-(5-methylpyridin-2-yl)ethanamine. (96% crude yield)

When evaporating the ether, the water bath temperature was kept at 25°C. since the boiling point of the amine is probably around 100° C.

Step 6: A mixture of 2 g of (2-methoxy-4-methylphenyl)methanamine (fromstep 3) and Et₃N (3.7 mL, 2 eq) in dry CH₃CN (45 mL) was cooled to 0° C.under N₂ atmosphere and ethyl 2-chloro-2-oxoacetate (1.47 mL, 1 eq) wasadded dropwise. After the addition was complete, the reaction mixturewas stirred at room temperature for 4 hours and2-(5-methylpyridin-2-yl)ethanamine (2.52 g, 1.4 eq, from step 5) wasadded. The reaction was heated at reflux for 24 hours. After cooling thesolvent was removed under reduced pressure and the residue was dissolvedin ethyl acetate and washed successively with water and brine, driedover MgSO₄, filtered and evaporated. The residue was chromatographed onsilica gel (eluent: 25-35% acetone in hexane) and recrystallized fromethyl acetate/hexane and ethanol/water to give 650 mg ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide(15%).

Example 30-1 Improved Preparation and Purification ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide

N¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamidewas prepared according to the synthetic strategy shown in Scheme 1. Thefollowing synthetic description makes reference to the compoundnumbering shown in this scheme.

Step 1—Preparation of 2-methoxy-4-methyl-benzamide

A mixture of 2-hydroxy-4-methyl-benzamide (15.1 g, 0.1 mol) and K₂CO₃(41.7 g, 0.3 mol) in 1000 mL of acetone was heated to reflux for 1 hourbefore Me₂SO₄ was added at that temperature. The resulting mixture wasrefluxed overnight. The reaction mixture was then filtered and thefiltrate was concentrated. The resulting residue was dissolved in 1000mL of methanol/ammonia (1:1) and stirred at room temperature for 3hours. After removal of the solvent, the residue was recrystallized fromEtOAc/PE to afford 102 g of 2-methoxy-4-methyl-benzamide (yield 61%). ¹HNMR (400 MHz, DMSO-d₆): δ 7.72 (d, J=7.6 Hz, 1H), 7.57 (br s, 1H), 7.46(br s, 1H), 6.92 (s, 1H), 6.81 (d, J=7.6 Hz, 1H), 3.85 (s, 3H), 2.31 (s,3H).

Step 2—Preparation of 2-methoxy-4-methyl-benzylamine hydrochloride salt

To a solution of 2-methoxy-4-methyl-benzamide (41 g, 0.25 mol) in 1500mL of THF was added lithium aluminum hydride (19 g, 0.5 mol) at 0° C.under N₂ atmosphere. The resulting mixture was heated to refluxovernight. After cooling, the reaction mixture was quenched by 10% NaOHaqueous solution. The reaction mixture was filtered and the filtrate wasconcentrated to the crude product. The residue was dissolved in Et₂O andthen added HCl/Et₂O solution. The precipitate was collected and washedwith TMBE to afford the segment A in Scheme 1 (35 g, yield 75%). ¹H NMR(300 MHz, DMSO-d₆): δ 8.15 (br s, 3H), 7.25 (d, J=7.5 Hz, 1H), 6.88 (s,1H), 6.77 (d, J=7.5 Hz, 1H), 3.94-3.86 (m, 2H), 3.79 (s, 3H), 2.30 (s,3H).

Step 3—Preparation of 2-(5-methylpyridin-2-yl)acetonitrile

To a solution of anhydrous acetonitrile (10.1 mL, 191.83 mmol, 3.3equiv.) in dry THF (500 mL) was added dropwise n-butyl lithium (2.5 M inhexane, 69.8 mL, 174.39 mmol, 3 equiv.) at minus 78° C. under a nitrogenatmosphere. The resulting white suspension was stirred at minus 78° C.for 1 hour, and then a solution of 2-bromo-5-methylpyridine (10.0 g,58.13 mmol, 1 equiv.) in dry THF (30 mL) was added. The reaction mixturewas kept at minus 78° C. for 1 hour and then warmed up slowly to roomtemperature and stirred for another hour. Ice/water was added and thelayer was separated. The organic layer was washed with water and brine,dried over MgSO₄, filtered, and evaporated to give 18 g of crudeproduct, which was used for next step reaction without furtherpurification.

Step 4—Preparation of 2-(5-methylpyridin-2-yl)ethamine hydrochloridesalt

To a solution of 18 g of crude 2-(5-methylpyridin-2-yl)acetonitrile indry THF (100 mL) was added dropwise borane-dimethylsulfide complex (10 Min THF, 23.2 mL, 232.5 mmol, 4 equiv.) at 0° C. under nitrogenatmosphere. The reaction mixture was then heated to 60° C. overnight.The reaction was cooled to room temperature, aqueous HCl (6 N, 40 mL)was added carefully, and the reaction mixture was heated at 70° C. for 2hours. After cooling, water was added and the resulting solution waswashed with ether. The aqueous layer was basified with aqueous NaOH (10N) at 0° C. and saturated with K₂CO₃, followed by extraction with ether(5×100 mL). The organic layer was dried over MgSO₄, filtered, andevaporated to give 7.6 g of crude product. The crude product wasdissolved in CH₂Cl₂ and treated with HCl/Et₂O to give2-(5-methylpyridin-2-yl)ethamine hydrochloride salt (4 g, yield 40%based on compound 3), which was used for the next step reaction withoutfurther purification.

Step 5—Preparation ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide (one pot)

To a mixture of 1.8 g of (2-methoxy-4-methylphenyl)methanaminehydrochloride salt and triethylamine (5.1 mL, 3 equiv.) in dryacetonitrile (45 mL) was added ethyl 2-chloro-2-oxoacetate (1.47 mL, 1equiv.) dropwise at 0° C. under nitrogen atmosphere. After the additionwas complete, the reaction mixture was stirred at room temperature for 2hours and 2-(5-methylpyridin-2-yl)ethanamine (2.52 g, 1.4 equiv.) wasadded. The reaction was heated at reflux for 24 hours. After removal ofthe solvent, the residue was dissolved in ethyl acetate and washedsuccessively with water and brine, dried over MgSO₄, filtered, andevaporated. The residue was washed with methyl t-butyl ether (20 mL) andrecrystallized from ethanol to give 1.3 g ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide as a white powder (yield 30%).

m/e=342 [M+1]; m.p.=133.5-134° C.; ¹H NMR (400 MHz, CDCl₃): δ 8.38 (s,1H), 8.07 (br s, 1H), 7.85 (br s, 1H), 7.40 (dd, J₁=1.6 Hz, J₂=8 Hz,1H), 7.12 (d, J=7.6 Hz, 1H), 7.03 (d, J=7.6 Hz, 1H), 6.71 (d, J=7.2 Hz,1H), 6.68 (s, 1H), 4.42 (d, J=6.4 Hz, 2H), 3.84 (s, 3H), 3.71 (q, J=6.4Hz, 2H), 2.97 (t, J=6.4 Hz, 2H), 2.33 (s, 3H), 2.30 (s, 3H); ¹³C NMR(400 MHz, CDCl₃): 18.6, 22.2, 36.9, 39.4, 39.9, 55.8, 111.9, 121.6,122.6, 123.3, 130.2, 131.6, 137.7, 139.9, 150.5, 156.1, 158.1, 160.0,160.5; Elemental Analysis: Calculated for C₁8H₂₁N₃O₃.¼H₂O: C, 65.97; H,6.85; N, 12.15. Found: C, 66.10; H, 7.34; N, 12.17.

Example 30-2 Alternative Improved Preparation and Purification ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide

N¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamidewas also prepared according to the synthetic strategy shown in Scheme 2.The following synthetic description makes reference to the compoundnumbering shown in this scheme.

2-methoxy-4-methyl-benzamid and 2-methoxy-4-methyl-benzylaminehydrochloride salt were prepared as described above.

Step 3—Preparation of 2-(5-methylpyridin-2-yl)acetonitrile

To a solution of anhydrous acetonitrile (10.1 mL, 191.83 mmol, 3.3equiv.) in dry THF (500 mL) was added dropwise n-butyl lithium (2.5 M inhexane, 69.8 mL, 174.39 mmol, 3 equiv.) at minus 78° C. under nitrogenatmosphere. The resulting white suspension was stirred at minus 78° C.for 1 hour, and then a solution of 2-bromo-5-methylpyridine (10.0 g,58.13 mmol, 1 equiv.) in dry THF (30 mL) was added. The reaction mixturewas kept at minus 78° C. for 1 hour and then warmed up slowly to roomtemperature and stirred for another hour. Ice water was added and thelayer was separated. The organic layer was washed with water and brine,dried over MgSO₄, filtered, and evaporated to give 18 g of crudeproduct. The crude product was purified by silica-gel columnchromatography (eluent, PE/EtOAc=15:1) to give2-(5-methylpyridin-2-yl)acetonitrile (6.2 g, yield 80%). ¹H NMR (300MHz, CDCl₃): δ 8.40 (d, J=3.0 Hz, 1H), 7.54 (dd, Ji=3.0 Hz, J₂=6.0 Hz,1H), 7.32 (d, J₂=6.0 Hz, 1H), 3.90 (s, 2H), 2.40 (s, 3H).

Step 4—Preparation of 2-(5-methylpyridin-2-yl)ethamine

To a solution of crude 2-(5-methylpyridin-2-yl)acetonitrile (3 g, 22.7mmol) in saturated NH₃-MeOH (50 mL) was added 0.5 g of Raney Ni. Thereaction mixture was then hydrogenated under 6 MPa at room temperatureovernight. The reaction mixture was filtered and the filtrate wasconcentrated in vacuo to afford 2-(5-methylpyridin-2-yl)ethamine (3.02g, yield 97%), which was used without further purification. Noimpurities were detected by ¹H-NMR. ¹H NMR (400 MHz, DMSO-d₆): δ 8.27(br s, 1H), 7.46 (d, J=8.4 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H), 2.69-2.83(m, 4H), 2.21 (s, 3H).

Step 5—Preparation of ethyl2-(2-methoxy-4-methylbenzylamino)-2-oxoacetate

To a solution of 2-methoxy-4-methylbenzylamine hydrochloride salt (10 g,0.053 mol) and triethylamine (30 g) in dry acetonitrile (100 mL) wasadded ethyl 2-chloro-2-oxoacetate (7.28 g, 0.053 mol) dropwise at 0° C.under nitrogen atmosphere. After the addition was completed, thereaction mixture was stirred at room temperature for 4 hours. Thesolvents were removed in vacuo and the residue was dissolved in ethylacetate, washed with brine (100 mLX×3), dried with Na₂SO₄, and thesolvents were removed to afford the title compound 5 (12 g, yield 89%),which was used for next step reaction without further purification. Noimpurities were detected by ¹H-NMR. ¹H NMR (400 MHz, DMSO-d₆): δ 9.10(br s, 1H), 6.98 (d, J=8 Hz, 1H), 6.79 (s, 1H), 6.70 (d, J=8 Hz, 1H),4.19-4.27 (m, 4H), 3.77 (s, 3H), 2.26 (s, 3H), 1.26 (t, J=7.2 Hz, 3H).

Step 6—Preparation ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide

To a mixture of compound 5 (36.9 g, 0.147 mol) and Segment B (30 g, 0.22mol) was added triethylamine (120 mL) and dry acetonitrile (800 mL). Thereaction mixture was heated to reflux for 34 hours. The solvents wereremoved by evaporation in vacuo and the residue was dissolved in ethylacetate (1 L), washed with water (300 mL×3), and dried with Na₂SO₄.After filtration and removal of solvents in vacuo, the residue wasrecrystallized from ethanol/water (10:1) to give 17 g ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide (yield 44%). ¹H NMR (300 MHz, DMSO-d₆): δ 8.83 (m, 2H), 8.30(d, J=2.1 Hz, 1H), 7.49 (dd, J₁=2.1 Hz, J₂=8.4 Hz, 1H), 7.20 (d, J=7.8Hz, 1H), 6.91 (d, J=7.5 Hz, 1H), 6.79 (s, 1H), 6.68 (d, J=7.5 Hz, 1H),4.23 (d, J=6 Hz, 2H), 3.77 (s, 3H), 3.43-3.50 (q, J₁=7.5 Hz, J₂=14.4 Hz,2H), 2.89 (t, J=7.5 Hz, 2H), 2.23 (s, 3H), 2.26 (s, 3H).

This method resulted in an improved yield of 85% when compared to theprevious example (40%) yield by purification of compound 4 with columnchromatography and by using H₂/Raney Ni instead of BH₃—SMe₂. Further,the one-pot preparation ofN¹-(2-methoxy-4-methylbenzyl)-N²-(2-(5-methylpyridin-2-yl)ethyl)oxalamide shown in the previous example, was divided into two steps inthis example. The yield was improved to 44% by purification of2-(2-methoxy-4-methylbenzylamino)-2-oxoacetate in this example, from 30%yield without any purification of2-(2-methoxy-4-methylbenzylamino)-2-oxoacetate in the one potpreparation described in the previous example.

Umami/Savory Flavor Experiments Using Human Panelists:

General Panelist Selection: Basic screening of sensory taste testers:Potential panelists were tested for their abilities to rank and rateintensities of solutions representing the five basic tastes. Panelistsranked and rated intensity of five different concentrations of each ofthe five following compounds: sucrose (sweet), sodium chloride (salty),citric acid (sour), caffeine (bitter), and monosodium glutamate(savory). In order to be selected for participation in testing,panelists needed to correctly rank and rate samples for intensity, witha reasonable number of errors.

Preliminary Taste Tests: The panelists selected in the above procedurewere deemed qualified for performing Preliminary Taste Testingprocedures. The preliminary taste tests are used to evaluate newcompounds for intensity of basic tastes and off-tastes. A small group ofpanelists (n=5) taste approximately 5 concentrations of the compound(range typically between 1-100 μM, in half-log cycles, e.g., 1, 3, 10,30, and 100 μM) in water and in a solution of 12 mM MSG to evaluateenhancement. Panelists rate the five basic tastes (sweet, salty, sour,bitter, and savory) as well as off-tastes (such as chemical, metallic,sulfur) on a labeled magnitude scale. Samples are served in 10 mLportions at room temperature. The purpose of the test is to determinethe highest concentration at which there is no objectionable off-taste,and determine if obvious savory taste or enhancement of savory tasteexists at any of the concentrations tested.

If the compound is effective and does not have objectionable off-tastes,it is tested with a trained (expert panel) in a larger study.

Trained Panelist Selection: A trained expert panel was used to furtherevaluate compounds that had been tested with the preliminary taste test.

Panelists for the trained panel were selected from the larger group ofqualifying taste panelists. Panelists were further trained on savorytaste by ranking and rating experiments using MSG and IMP combinations.Panelists completed a series of ranking, rating, and difference fromreference tests with savory solutions. In ranking and ratingexperiments, panelists evaluated easy MSG concentrations (0, 6, 18, 36mM) and more difficult MSG concentrations (3, 6, 12, 18 mM MSG) inwater.

Compound testing with Trained Panel: Compounds tested by the trainedpanel were evaluated in difference from reference experiments. Panelistswere given a reference sample (12 mM MSG+100 μM IMP) and asked to ratesamples on a scale of −5 to +5 in terms of difference in savory tastefrom the reference (score: −5=much less savory taste than the reference;0=same savory taste as the reference; +5=much more savory taste than thereference). Test samples were solutions with varying amounts of MSG,IMP, and the compound. Typically, each session compares the referencesample to numerous test samples. Tests typically included varioussamples with varying concentrations of MSG and IMP, as well as one blindsample of the reference itself, to evaluate panel accuracy. Results ofthe taste tests are describe in table 3 and shows that compounds of theinvention have been found to provide savory taste or enhancement of thesavory taste at 3 μM+MSG when compared to 100 μM IMP+MSG. Compounds weretested against the reference in samples with and without 12 mM MSG. Allsamples were presented in 10 ml volumes at room temperature. Twosessions were completed for each compound tested to evaluate panelreproducibility.

Taste Test in Product Prototype: could be done similarly as describedabove.

TABLE 3 Savory Taste Test Results Compound No. Chemical Name Taste DataExample 1 N-(heptan-4- 12 mM MSG + 3 μM cpd asyl)benzo[d][1,3]dioxole-5- strong as 12 mM MSG + carboxamide 100 μM IMPExample 5 (R)-methyl 2-(benzo[d][1,3] 12 mM MSG + 10 μM cpd asdioxole-6-carboxamido)-4- strong as 12 mM MSG + methylpentanoate 100 μMIMP Example 18 (R)-N-(1-methoxy-4- 12 mM MSG + 3 μM cpd asmethylpentan-2-yl)-3,4- strong as 12 mM MSG + dimethylbenzamide 100 μMIMP Example 19 (R)-methyl-2-(2,3- 12 mM MSG + 10 μM cpd asdimethylfuran-5- strong as 12 mM MSG + carboxamido)-4- 100 μM IMPmethylpentanoate Example 20 4-Methoxy-N-(1- 12 mM MSG + 3 μM cpd asmethoxymethyl-3-methyl- strong as 12 mM MSG + butyl)-3 -methyl-benzamide100 μM IMP Example 24 N-(2,4-Dimethoxy-benzyl)- 12 mM MSG + 1 μM cpd asN′-(2-pyridin-2-yl-ethyl)- strong as 12 mM MSG + oxalamide 100 μM IMP 1μM cpd as strong as 12 mM MSG Example 26 N-(2methoxy-4- 12 mM MSG + 1 μMcpd as methylbenzyl)-N′-(2(5- strong as 12 mM MSG + methylpyridin-2- 100μM IMP yl)ethyl)oxalamide 1 μM cpd as strong as 12 mM MSG Example 30N¹-(2-methoxy-4- 12 mM MSG + 0.3 μM cpd as methylbenzy1)-N²-(2-(5-strong as 12 mM MSG + methylpyridin-2- 100 μM IMP yl)ethyl)oxalamide 0.3μM cpd as strong as 12 mM MSG Example 22 (S)-N-(2,3-Dihydro-1H- 12 mMMSG + 1 μM cpd as inden-1-yl)-4-methoxy-3- strong as 12 mM MSG +methylbenzamide 100 μM IMP 1 μM cpd as strong as 12 mM MSG

Example 31 Soup Preparation Using an Ethanol Stock Solution

A compound from Table 3 is diluted using 200 proof ethanol to 1000× thedesired concentration in soup. The compound can be sonicated and heatedto ensure complete solubility in ethanol. The soup from bouillon base ismade by adding 6 g of vegetable bouillon base in 500 mL of hot water ina glass or stoneware bowl. The water is heated to 80° C. Theconcentration of MSG in the dissolved bouillon is 2.2 g/L and there isno IMP added. After the bouillon base is dissolved, the ethanol stocksolution is added to the soup base. For 500 mL of soup, 0.5 mL of the1000× ethanol stock is added for a final ethanol concentration of 0.1%.If the ethanol interferes with the taste of the soup, a higherconcentration of ethanol stock solution can be prepared provided thecompound is soluble.

Example 32 Chip Preparation

A salt mixture of a compound of the invention is made by mixing withsalt such that a 1.4% of the salt mixture added w/w to chips wouldresult in the desired concentration of the compound. For 1 ppm final ofthe compound on chips, 7 mg of the compound is mixed with 10 g of salt.The compound is ground using a mortar and pestle with the salt and thecompound and salt are mixed well. The chips are broken into uniformsmall pieces by using a blender. For each 98.6 g of chips, 1.4 g of thesalt mixture is weighed out. The chip pieces are first heated in amicrowave for 50 seconds or until warm. The pieces are spread out on alarge piece of aluminum foil. The salt mixture is spread evenly over thechips. The chips are then placed in a plastic bag making sure that allthe salt is place in the bag as well. The salt mixture and chips arethen shaken to ensure that the salt is spread evenly over the chips.

Example 33 Spicy Tomato Juice or Bloody Mary Mix

A compound of the invention is added as a dry ingredient to a spiceblend, which may optionally include monosodium glutamate, and blendedthoroughly. Spice blend is dispersed into a portion of tomato paste,blended, and that blended paste is further blended into the remainingpaste. The paste is then diluted with water to make spicy tomato juiceor Bloody Mary mix.

Example 34 Human Taste Tests of Low Sodium Tomato Juice

Human taste tests were conducted in order to evaluate the ability of thecompounds of the invention to enhance the savory flavor of low sodiumtomato juice (which naturally comprises some monosodium glutamate).

Sample Preparation Procedure

The final tomato juice samples for taste testing were prepared so as tocomprise 90% (by volume) pre-made low sodium tomato juice stock (pH 4.2,80˜100 mg Na/8 oz, 16 mM of naturally occurring MSG), 5% (by volume) ofstock solutions formulated to produce selected final levels of sodium offinal juice, and 5% (by volume) of a stock solution of the compound ofthe invention. Selected oxalamide compounds of the invention weredissolved in LSB (low sodium phosphate buffer), to provide a stocksolution at 20 times the desired final concentration in the final tomatojuice. The desired final sodium concentration of the final tomato juicewas most experiments 73.6 mM (400 mg sodium in 8 oz. of juice),therefore a stock solution of NaCl was made at 1.48 M NaCl. The pH forthe stock solutions was adjusted to 4.2 using a 1 M citric acidsolution, and the stock solutions were sonicated to ensure the additivecompounds were completely dissolved. To produce a 1,000 mL final sampleof tomato juice sample for taste testing, 50 mL of the test compoundstock solution, and 50 mL of the sodium chloride were added to 900 mL ofthe pre-made low sodium tomato juice stock.

Human Taste Tests

Sixteen human subjects were used in the taste testing. The subjectsrefrained from eating or drinking (except water) for at least 1 hourprior to the test. Subjects ate a cracker and rinsed with water tocleanse the mouth before the start of the test. 15 mL samples wereserved in 2 oz. sample cups at room temperature. Panelists rinsed withwater between samples, and were encouraged to eat a cracker to removeall tastes before moving to the next sample. Samples were presented inrandomized counterbalanced order within each tasting session (withdifferent blinding codes). The panelists were asked to evaluateumaminess (savory level) make comments on the samples on an unstructuredline scale (scoring 0-10), in duplicate sessions. There were 5 minutesbreaks between tasting sessions, and a total of 4 sessions over a 2 dayperiod. The samples tasted are given below

Samples Tasted 400 mg Na/8 oz tomato juice 400 mg Na + 3 uM Compound24/8 oz tomato juice 400 mg Na + 3 uM Compound 30/8 oz tomato juice

Scores were averaged across panelists and sessions, and evaluated usinga 2-way ANOVA (factors: panelists and samples) and Duncan's multiplecomparison test (alpha=0.05) to determine significant differences inintensity ratings. Results are summarized below.

TABLE G Tomato Juice Taste Test Results Compound Chemical Name TasteData 24 N1-(2,4-dimethoxybenzyl) 3 μM cpd enhanced theN2-(2-(pyridin-2-yl)ethyl) savory taste of 16 mM oxalamide glutamate(naturally existing) in low sodium tomato juice by 1.4 to 1.5-fold 30N1-(2-methoxy-4- 3 μM cpd enhanced the methylbenzyl)- savory taste of 16mM N2-(2-(5-methylpyridin-2- glutamate (naturally existing)yl)ethyl)oxalamide in low sodium tomato juice by 1.8 to 1.9-fold

Example 35 Preparation of Savory Flavor Concentrate Compositions bySpray Drying

As described elsewhere herein, the savory compounds described herein areextremely potent; and it is desirable to prepare diluted flavorantconcentrate compositions in order to suitably formulate seasoning mixesand/or final comestible compositions. The spray-drying proceduresdescribed below were used to prepare two such solid flavorantconcentrate compositions, one comprising 0.10% (w/w) of the amidecompounds of Example 1 and the other comprising the oxalamide compoundof Example 24.

344.74 grams of commercially available Neobee M-5 medium chaintriglyceride composition was placed in a 2 liter stainless steelcylinder container and heated to 200 F on a steam table, with agitationby a Lightnin Mixer. 18.14 grams of the amide compound of Example 1(Compound 1) was slowly added with high agitation and mixed for 30minutes until the compound was thoroughly dissolved and the solution wasclear, to form a savory pre-blend composition, then the temperature waslowered to 180° F.

In a separate 2 liter stainless steel cylinder container, 2721.60 g ofwater was stirred with a Lightnin mixer and heated to a temperature of140° F., then the Lightnin Mixer speed was set to 500 rpm to create avortex, and 1451.52 g of N-Lok 1930 modified food starch was slowlyadded, and mixed for 20 minutes. Then 1814.40 g of the savory pre-blendcomposition described above was slowly added to the aqueous N-loksolution and mixed for 30 minutes, at 500 rpms, and the temperaturereduced to 120° F., then the mixture was homogenized using a Gaulinhomogenizer, Model E, with a first stage set at 2000 psi and a secondstage set at 500 psi, to form an emulsion.

The resulting emulsion was spray dried using a Stock-Bowen ConicalLaboratory Spray-Aire spray dryer with an inlet temperature of 203° C.,outlet temperature of 119° C. with a flow rate of 160 ml/minute and anair pressure of 40 psig, to form 1306.20 grams of a powder comprising 1%of the compound of Example 1. This was equal to a 71.99% yield, or a18.01% loss of product in the evacuation chamber. One pound (453.6 g) ofthe spray dried powder was combined with 9.0 lbs (4082.4 g) of 10DEmaltodextrin (Tate and Lyle) in a 40 quart Hobart planetary mixer andmixed for 20 minutes, to create a dry savory flavor concentrate powderhaving 0.10% (wt/wt) of the compound of Example 1. HPLC analysisindicated 82% of the theoretical amount of Compound 1. An equivalentprocessing procedure was used to prepare a savory flavor concentratecomposition comprising 0.1% (wt/wt) of the compound of Example 24(Compound 24). In particular, 349.272 g of Neobee M-5 and 13.608 g ofthe oxalamide compound of Example 24 were mixed at 200° F. untildissolved to form a savory pre-blend composition, and the temperature ofthe solution was lowered to 180° F.

1451.52 g of N-Lok 1930 and 2721.60 g of water was mixed and heateduntil it reached a temperature of 140° F., the a vortex was createdusing a Lightnin Mixer at 500 rpms and 1814.40 g of the savory pre-blendcomposition and mixed for 30″ at 500 rpms, then the solution was held at120° F. and homogenized using a Gaulin, model E, homogenizer. Theresulting emulsion was spray dried with an inlet temperature of 203° C.,outlet temperature of 119° C., with a flow rate of 160 ml/minute and anair pressure of 40 psi, to produce 1400.72 grams of spray dried powderhaving 0.75% (wt/wt) of the compound of Example 24.

1.334 lbs (605.1024 g) of the spray dried powder was mixed with 8.666lbs (3930.8976 g) of a 10 DE maltodextrin (Tate and Lyle) to produce afinal dry savory flavor concentrate powder having 0.10% (wt/wt) of thecompound of Example 24.

Example 36 Replacement of MSG in Sausage Seasonings

Summary:

The savory flavor concentrates compositions comprising the compounds ofExamples 1 or 24 were used to prepare sausage seasoning mixes forItalian sausage, and example sausages themselves, and compared in humantaste tests to control sausages both with and without MSG. Human tasterscompared the samples and reported that the sausage comprising both ofthe compounds of Examples 1 and 24 had a more savory flavor than thecontrol sausages, perceived increased salty flavor, and increased spicytastes as compared with the control sausages.

Sausage Preparations:

Three different dry Italian sausage seasoning mixes were formulated bydry blending the ingredients shown in the Tables below, then rehydratedduring the formulation of corresponding sausages. One control sample ofseasoning mix (Tables 36-1a, b) contained no MSG, and no compounds ofthe invention. A second sample of seasoning mix and sausage containedadded MSG (Tables 36-2a, b). A third sample of Seasoning mix andcorresponding sausage also contained added savory flavor concentratecompositions (prepared as per Example 34) containing (0.1% wt/wt) ofcompounds 1 and 24 (Tables 36-1a, b).

TABLE 36-1a Italian sausage seasoning mix without MSG Formula %Ingredient Grams (wt/wt) Salt (Morton Salt) 64.16 32.08 Sugar (C&H#801461) 37.74 18.87 Paprika (Pacific Spice SA #1406) 37.74 18.87 Garlicpowder (American Ingredients #5557) 30.56 15.28 Fennel Seeds Toasted(Spice Island 6C08B ) 11.32 5.66 Black pepper 28 mesh (Pacific Spice SA9.44 4.72 #01406) Parsley flakes (Pacific Spice SA #C1406) 3.78 1.89Ground Anise seed (retail Kroger 09BK) 3.38 1.69 Cayenne pepper (PacificSpice SA#C1406) 1.88 0.94 TOTAL 200 100 Analysis of 10% solution of thedry mix pH 5.00 % Salt 31.53 Brix 6.60

TABLE 36-1b Sausage Preparation (No MSG) Formula % Ingredient Grams(wt/wt) Pork, ground 20% fat 280.68 93.56 Dry mix Italian sausageseasoning 15.9 5.30 without MSG Water 3.42 1.14 TOTAL 300 100

TABLE 36-2a Italian sausage seasoning mix with MSG Formula % IngredientGrams (wt/wt) Salt (Morton Salt) 62.96 31.48 Monosodium glutamate(Ajinomoto #310704) 3.70 1.85 Sugar (C&H #801461) 37.04 18.52 Paprika(Pacific Spice SA #1406) 37.04 18.52 Garlic powder (American Ingredients#5557) 30.00 15.00 Fennel Seeds Toasted (Spice Island 6C08B) 11.12 5.56Black pepper 28 mesh (Pacific Spice SA 9.26 4.63 #01406) Parsley flakes(Pacific Spice SA #C1406) 3.70 1.85 Ground Anise seed (retail Kroger09BK) 3.32 1.66 Cayenne pepper (Pacific Spice SA#C1406) 1.86 0.93 TOTAL200 100 Analysis of 10% solution of the dry mix pH 5.00 % Salt 31.53Brix 6.60

TABLE 36-2b Sausage Preparation (with MSG) Formula % Ingredient Grams(wt/wt) Pork, ground (fat level 20%) 280.41 93.47 Dry mix Italiansausage seasoning 16.17 5.39 with MSG Water 3.42 1.14 TOTAL 300 100

TABLE 36-3a Italian sausage seasoning mix with 0.5 ppm of Compound 1 and0.7 ppm of Compound 24. Formula % Ingredient Grams (wt/wt) Salt (MortonSalt) 61.60 31.37 Flavor concentrate comprising 0.10% 1.82 0.92 Compound1 Flavor concentrate comprising 0.10% 2.54 1.29 Compound 24 Sugar (C&H#801461) 36.24 18.45 Paprika (Pacific Spice SA #1406) 36.24 18.45 Garlicpowder (American Ingredients #5557) 29.34 14.95 Fennel Seeds Toasted(Spice Island 6C08B) 10.88 5.54 Black pepper 28 mesh (Pacific Spice SA9.06 4.61 #01406) Parsley flakes (Pacific Spice SA #01406) 3.62 1.85Ground Anise seed (retail Kroger 09BK) 3.26 1.67 Cayenne pepper (PacificSpice SA#C1406) 1.82 0.92 TOTAL 200 100 Analysis of 10% solution of thedry mix pH 5.00 % Salt 31.53 Brix 6.60

TABLE 36-3b Sausage Preparation Formula % Ingredient Grams (wt/wt) Pork,ground 20% fat 280.35 93.45 Dry mix Italian sausage with Compounds 16.235.41 1 (9 ppm) and 24 (13 ppm) Water 3.42 1.14 TOTAL 300 100Sensory Evaluations:

Ten trained tasters were trained to identify the relative intensities ofthe following taste attributes: salty, savory/meaty, umami, brothiness,bitter, linger and off flavors. A rank-rating intensity test was used toevaluate the Italian Sausagesamples. A rank rating is a difference testto determine whether a sensory difference is perceived between varioussamples. Samples that were evaluated were the Italian Sausage no MSG,Italian sausage with MSG, Italian sausage with compounds 1 and 24 but noMSG. The panelists evaluated 3 randomized, blind coded samples of oneounce of Italian sausage served at 140° F., and ranked the intensity ofsavory, salt, spice flavors on a scale of 0-10: 0 being the leastintense and 10 being the most intenese, and the scores were averaged forindividual category.

TABLE 36c Average scores of individual category for Italian SausageSavory Spice Salt With MSG 6.13 5.42 5.24 Without MSG 4.70 5.88 5.56With Compounds 1 and 24 6.34 6.42 5.97

Conclusions: The panelists were able to detect differences between thesamples.

-   -   a. Savory: panelists ranked samples without MSG to be the least        savory and samples with savory enhancer compounds 1 and 24 to be        the most savory    -   b. Salt: panelist ranked samples with MSG to be the least salty        and samples with savory enhancer compounds 1 and 24 to be the        most salty    -   c. Spice flavor: panelists ranked samples with MSG to have the        least spice flavor and samples with savory enhancer compounds 1        and 24 to have the most spice flavor

Example 37 Partial replacement of MSG in Ramen Noodle Soup

Summary:

A mixture of the compounds of Examples 1 and 24 were used tore-formulate Ramen Noodle Soup to have only ⅓ of a control level of MSG,and compared via human taste tests to a control soup having a fullloading of MSG, and another control soup having only ⅓ of a normalloading of MSG. Human tasters could distinguish between the savoryflavor of a control soup comprising a full loading of MSG, and a controlsoup with only ⅓ of the MSG, but could not distinguish the control soupwith full MSG loading from the soup re-formulated with ⅓ of the MSG andthe two compounds of the invention.

Noodle Soup Sample Preparation Procedures;

Ramen Noodle Soup Preparations: The procedures below were used toprepare three variations of Ramen Noodle Soup Seasoning blends, whichwere added to cooked Ramen soup, for human taste testing. One Controlseasoning blend comprised a full loading of MSG (Tables 1a, 1b, and 1c).A second Control seasoning blend comprised a ⅓ loading of MSG (Tables2a, 2b, and 2c). A third seasoning blend comprised the flavorconcentrate compositions comprising compounds 1 and 24, and a ⅓ loadingof MSG.

Ramen Noodle Soup Seasoning Mixes:

The dry ingredients listed in Tables 37-1a,37-2a,37-3a were dry blendedin a 6 quart bowl using a kitchen aid blender with a paddle attachmentfor 10 minutes and then hand blended with a rubber spatula to ensurevisual homogeneity. The dry seasoning mixes were stored in apolyurethane non-vented food bag prior to soup preparation. Theseasonings were then used to prepare Ramen Noodle Soup using hot wateraccording to the proportions in Tables 37-1c,37-2c,37-3c. Theproportions of ingredients used to prepare Ramen Noodle Soup actuallyhaving Noodles are illustrated in Tables 37-1c,37-2c,37-3c, but thosesamples were not actually tasted.

TABLE 37-1a Ramen Noodle Soup Seasoning Mix (full MSG)- Control FormulaA Formula % Ingredient (wt/wt) Salt (Morton Salt) 31.53 MonosodiumGlutamate (Ajinomoto) 15.24 Autolyzed Yeast Extract (Sensient #945) 0.79Hydrolyzed Vegetable Protein (Basic Food 5.25 Flavors #C-303) AjitideI + G (Ajinomoto) 0.26 Soy Sauce Powder (Nikken #5310) 11.04 GarlicPowder (American Food Flavors, 9.25 Inc #GA45) Powdered Chicken(Henningson 9.04 C-100-1B-AR #730) Chicken Flavor (Mastertaste #F42X32)5.78 Onion Powder (Elite Spice #516) 4.20 Sugar, Granulated (C & H) 2.57Powdered Chicken Fat (Henningson #732) 2.52 Tumeric (McCormick) 1.05White Pepper (Pacific Natural Spices) 0.63 Celery Powder (Food Source,Inc. #60) 0.53 Citric Acid (ADM) 0.32 TOTAL 100.00%

TABLE 37-1b Preparation of Ramen Noodle Soup(as prepared with noodles)Formula % Ingredient Grams (wt/wt) Ramen Noodle Soup Seasoning-Formula A8.99 1.61 Ramen Noodles (retail) 77.36 13.82 Water 473.20 84.57 TOTAL559.55 g 100.00%

TABLE 37-1c Preparation of Ramen Noodle Soup (as evaluated by the tastepanel) Formula % Ingredient Grams (wt/wt) Ramen Noodle SoupSeasoning-Formula A 17.98 1.86 Water 946.40 98.14 TOTAL 964.38 g 100.00%

TABLE 37-2a Ramen Noodle Soup Seasoning, 66% reduced MSG-Formula BFormula % Ingredient (wt/wt) Salt (Morton Salt) 35.07 MonosodiumGlutamate (Ajinomoto) 5.71 Hydrolyzed Vegetable Protein (Basic Food 5.85Flavors #C-303) Autolyzed Yeast Extract (#945) 0.88 Ajitide I + G(Ajinomoto) 0.29 Soy Sauce Powder (Nikken #5310) 12.28 Garlic Powder(American Food Flavors, Inc #GA45) 10.29 Powdered Chicken (HenningsonC-100-1B-AR #730) 10.05 Chicken Flavor (Mastertaste #F42X32) 6.43 OnionPowder (Elite Spice #516) 4.68 Sugar, Granulated (C & H) 2.86 PowderedChicken Fat (Henningson #732) 2.81 Tumeric (McCormick) 1.17 White Pepper(Pacific Natural Spices) 0.70 Celery Powder (Food Source, Inc. #60) 0.58Citric Acid (ADM) 0.35 TOTAL 100%

TABLE 37-2b Preparation of Ramen Noodle Soup (as prepared with noodles)Formula Ingredient Grams % (wt/wt) Ramen Noodle Soup Seasoning-Formula B  8.09  1.45 Ramen Noodles (retail)  77.36 13.85 Water 473.20 84.70TOTAL 558.65 g 100%

TABLE 37-2c Preparation of Ramen Noodle Soup as evaluated by the tastepanel) Formula Ingredient Grams % (wt/wt) Ramen Noodle SoupSeasoning-Formula B  16.18  1.68 Water 946.40 98.32 TOTAL 964.38 g 100%

TABLE 37-3a Ramen Noodle Soup Seasoning, 66% reduced MSG with savoryenhancers-Formula C Formula % Ingredient (wt/wt) Salt (Morton Salt)33.39  Flavor Concentrate with 0.1% Compound 24 (8.5 ppm) 0.85 FlavorConcentrate with 0.1% Compound 1 (39.5 Ppm 3.96 Monosodium Glutamate(Ajinomoto) 5.44 Hydrolyzed Vegetable Protein (Basic Food Flavors 5.56#C-303) Autolyzed Yeast Extract (#945) 0.83 Ajitide I + G (Ajinomoto)0.28 Soy Sauce Powder (Nikken #5310) 11.69  Garlic Powder (American FoodFlavors, Inc #GA45) 9.79 Powdered Chicken (Henningson C-100-1B-AR #730)9.57 Chicken Flavor (Mastertaste #F42X32) 6.12 Onion Powder (Elite Spice#516) 4.45 Sugar, Granulated (C & H) 2.73 Powdered Chicken Fat(Henningson #732) 2.67 Tumeric (McCormick) 1.11 White Pepper (PacificNatural Spices) 0.67 Celery Powder (Food Source, Inc. #60) 0.56 CitricAcid (ADM) 0.33 TOTAL 100%

TABLE 37-3b Preparation of Ramen Noodle Soup (as prepared with noodles)Formula Ingredient Grams % (wtlwt) Ramen Noodle Soup Seasoning-Formula C 8.49  1.52 Ramen Noodles (retail)  77.36 13.84 Water 473.20 84.64 TOTAL559.05 g 100%

TABLE 37-3c *Preparation of Ramen Noodle Soup (as evaluated by thesensory taste panel) Formula Ingredient Grams % (wt/wt) Ramen NoodleSoup Seasoning-Formula B  16.98  1.76 Water 946.40 98.24 TOTAL 968.38 g100% *Formula 37-3 a contained a combination of compounds 24 and 1 in aratio of 1:4.5 in the finished ramen soup that had a final concentrationlevel of 0.15 ppm of compound 24 and 0.70 ppm of compound 1 in thefinished ramen soup.Sensory Evaluation Procedures:

Eight trained external tasters were were trained to identify relativeintensities of the following taste attributes: salty, savory/meaty,umami, brothiness, bitter, linger and off flavors in paired comparisontests. Paired comparison tests attempt to determine whether aperceivable sensory difference exists between two blind coded randomizedsamples. Each sample contained 2 ounces of ramen soup, served at 120°F., and each panelist conducted a total of five paired comparison tests.

Evaluation #1: The Ability to Distinguish Savory Flavors of High and LowMSG Control Formulations

The objective was to determine whether panelists could tell thedifference in savory flavor between the high MSG control (Full MSG,Formula A) and the 66% reduced MSG Control (Formula B);

Results:

Eight panelists evaluated the 100% MSG Ramen broth verses the 33% MSGRamen broth in paired comparison tests five times. The results showedthat 25 evaluations rated the 100% MSG ramen broth as more savory thanthe 33% MSG ramen broth while 15 tests rated the 33% MSG ramen broth asmore savory. See Table 37-4 for results.

TABLE 37-4 100% MSG ramen broth versus 33% MSG ramen broth. n = 40 (8Panelists × 5 reps). Samples Totals 100% MSG ramen soup selected 25  33%MSG ramen soup selected 15 Total 40 Confidence 0.923 100% MSG ramen soupselected (p-value) 0.077

Conclusion: Panelists could differentiate between 100% MSG ramen soupversus the 66% reduced MSG ramen soup; and correctly identified the 100%MSG sampleto be significantly more savory than 33% MSG ramen broth.(p<0.1).

Evaluation #2—Comparison of High MSG Ramen Noodle Soup and aReformulated Soup Comprising Compounds 1 and 24 and 66% reduced MSG.

The objective was to determine whether panelists could tell thedifference in savory flavor between the high MSG noodle soup (Full MSG,Formula A) and the reformulated noodle soup comprising 66% reduced MSG,and compound 1 at 0.7 ppm and compound 2 at 0.15 ppm.

Results:

Eight panelists evaluated the 100% MSG ramen soup verses the 33% MSGramen broth plus 0.15 ppm Compound 24 and 0.7 ppm Compound 1 in pairedcomparison tests repeated five times to obtain an N=40. The resultsshowed that 20 evaluations rated the 100% MSG ramen soup as more savoryand 20 evaluations rated the 33% MSG+0.15 ppm Compound 24 and 0.70 ppmCompound 24 ramen soup as more savory. See Table 37-5 for results.

TABLE 37-5 100% MSG ramen soup versus 33% MSG + 0.15 ppm Compound 24 and0.7 ppm Compound 1 ramen soup. n = 40 (8 Panelists × 5 reps). SamplesTotals 100% MSG ramen soup selected 20 33% MSG + 0.15 ppm Compound 24and 20 0.7 ppm Compound 1 ramen broth selected Total 40 Confidence<0.125 100% MSG ramen soup selected (p-value) >0.875

Conclusion:

Panelists were unable to perceive a significant difference in savoryintensity between 100% MSG ramen noodle soup and 33% MSG with +0.15 ppmCompound 24 and 0.7 ppm Compound 1 ramen noodle soup (p<0.1). Therefore,MSG was significantly reduced and replaced with Senomyx's enhancer 5336and 5807 without any change in savory intensity.

Additional taste perceptions were reported by leasts some of thesubjects, including increased salt perception in the sample utilizingcompounds 1 and 24, and that the prolonged umami lingering effectsimproved the balance of flavor, and gave increased spice, herb and heatflavors in the sample utilizing compounds 1 and 24. Accordingly, use ofmixtures of compounds can allow for reduction MSG and other savorypotentiators such as IMP, GMP, etc, which may results in cost reduction.

Example 38 Replacement of MSG in Cajun BBQ Flavored Potato Chips

Summary:

Four samples of Cajun flavored potato chips were prepare and comparedvia human taste tests. One control chip sample had no MSG, anothercontrol chip sample had 0.6% MSG added, one chip sample had 3 ppm of theamide compound of Example 1 added in the form of a flavor concentratecomposition, and one chip sample had 3 ppm of the oxalamide compound ofExample 24 added in the form of a flavor concentrate composition. Inhuman focus group taste tests, nine of eleven panelists reported thatthe chip samples comprising the compounds of Examples 1 and 24 had anincreased intensity of Cajun spices and pepper flavors. Some panelistsreported that the chip samples comprising the compounds of Examples 1and 24 had an increased savory flavor.

Sample Preparations:

Two solid savory flavor concentrate compositions were prepared by spraydrying procedures equivalent to those reported in Example 35. One savoryflavor concentrate sample, comprising 0.01% (wt/wt) of the compound ofExample 1 was prepared via from the compound of Example 1, Neobee M-5medium chain triglycerides, N-Lok 1930 modified food starch, andmaltodextrin. Another savory flavor concentrate sample, comprising 0.01%(wt/wt) of the compound of Example 24 was similarly prepared via fromthe compound of Example 24, Neobee M-5 medium chain triglycerides, N-Lok1930 modified food starch, and maltodextrin.

A basic dry seasoning mix for providing Cajun flavor for the chips wasprepared by dry blending the ingredients in Table 38-1 using aKitchenAide blender for 5 minutes at 40-80 rpms using a wire whipattachment.

TABLE 38-1 Dry Seasoning Mix Formula A Formula % Ingredient Grams(wt/wt) Maltodextrin DE 18 (Grain Processing Co.) 29.33 29.33 Salt-Flour (Morton) 13.10 13.10 Honey Powder-50% honey (Mastertaste) 27.3727.37 Onion Powder (American Food Ingredients) 7.16 7.16 Garlic Powder(American Food Ingredients) 6.55 6.55 Paprika (McCormick) 2.18 2.18 RedPepper (McCormick) 1.09 1.09 Ground Cayane Red Pepper (McCormick) 0.710.71 White Pepper (McCormick) 0.22 0.22 Lime Powder (Mastertaste) 4.584.58 Ancho chili powder (McCormick) 0.95 0.95 Canola oil spray 0.87 0.87Tomato powder (McCormick) 5.62 5.62 Chardex Hickory MS (Red Arrow) 0.270.27 TOTAL 100.00 100.00

General Preparation of the Seasoned Chip Samples:

Kettle® Unsalted potato chips were hand broken into reasonably uniform½″×½″ pieces and placed in a polyethylene 26.7 cm×27.9 cm non-ventedfood bag. The chips were then heated in an Amana Commercial 1200 wattmicrowave for 20 seconds to release oils, and so the seasoning wouldadhere to the chips. Canola oil and was sprayed on the chips and thenthe various seasoning ingredients (the Dry Seasoning Mix describedabove, premixed with any added MSG or flavor concentrate compositions)were sprinkled into the bag. The bag was then shaken by hand for 1minute until all chips were evenly coated with the seasoning mixture(Table 2). Chips were allowed to cool to ambient temperature (20° C./68°F.) before tasting.

TABLE 38-2 Cajun BBQ Chips with no MSG or savory concentrate compostionsIngredient Grams Formula % (wt/wt) Kettle ® Chips- Unsalted 84.36 84.36Seasoning Mix A 7.36 7.36 MSG (Ajinomoto #310704) 0.18 0.18 Canola oilspray 8.10 8.10 TOTAL 100.00 100.00

TABLE 38-3 Cajun BBQ Chips with 0.6% MSG Ingredient Grams Formula %(wt/wt) Kettle ® Chips- Unsalted 83.67 83.67 Seasoning Mix A 7.63 7.63MSG (Ajinornoto #310704) 0.60 0.60 Canola oil spray 8.10 8.10 TOTAL100.00 100.00

TABLE 38-4 Cajun BBQ Chips- with 3 ppm Compound 1 Formula In!redientGrams % wt/wt Kettle ® Chips- Unsalted 81.09 81.09 Seasoning Mix A 7.637.63 Salt- Flour (Morton) 0.18 0.18 Camla oil spray 8.10 8.10 FlavorConcentrate Composition 3.00 3.00 containing 0.01% Compound 1 TOTAL100.00 100.00

TABLE 38-5 Cajun BBQ Chips- with 3 ppm Compound 24 Formula IngredientGrams % (wt/wt) Kettle ® Chips- Unsalted 81.09 81.09 Seasoning Mix A7.63 7.63 Salt- Flour (Morton) 0.18 0.18 Carola oil spray 8.10 8.10Flavor Concentrate Composition 3.00 3.00 containing 0.01% Compound 24TOTAL 100.00 100.00

Sensory Evaluation Procedures:

Eleven external tasters were trained to identify relative intensities ofthe following taste attributes: sweet, salty, savory/meaty, umami,brothiness, bitter, linger and off flavors. The panelists evaluated thefour sample types of Cajun BBQ chips in a Focus Group taste testprocedure and were instructed to write comments on several attributes:sweet, BBQ, Cajun spice, pepper, onion, salt, potato, umami, off tastes,other, and liking or disliking. And instructed to evaluate differencesin umami taste and possible enhancement of flavors and taste.

Results:

Overall, 9/11 panelists reported the samples containing Compounds 1 and24 to have the higher intensity of Cajun spice and pepper flavors.

Example 39 Replacement of MSG in Pizza Sauce

Summary:

A water soluble flavor concentrate composition comprising the compoundof Examples 1 was prepared and then used to replace MSG in pizza sauce,and compared via human taste tests to a pizza sauce having a fullloading of MSG. Most taste tasters could not distinguish between thesauces.

Water Soluble Flavor Concentrate Composition Comprising Compound 1:

For effective formulation in pizza sauce, a water soluble savory flavorconcentrate formulation comprising 0.1% of Compound 1 was developed bydissolution in propylene glycol.

18221.7600 g of propylene glycol (supplied by Gold Coast, Inc.) wasweighed out into a 2000 ml stainless steel cylinder, 18.2400 grams ofCompound 1 was slowly added to 700.00 g of the propylene glycol using aSilverson Mill (model: L4RT) having a square hole high shear screen andset at 2000 rpm, until all of the dry 5807 (Compound 1) was added to thepropylene glycol, the mill rpms was increased to 5,000-6,000 rpm's for8-10 minutes. The remaining 17,521.76 g of propylene glycol was placedinto a 10-gallon steam jacketed kettle equipped with a Lightnin Mixerutilizing a three blade impeller and heated to 50° C. (120° F.), thenthe milled solution was slowly added to the heated and agitatedpropylene glycol in the kettle. It took over 30 minutes of heating andmixing to completely solubilize Compound 1, then, the solution wasallowed to cool to 80° F., then a Unispense Filler was used to fillsterilized 2 oz. glass containers with 62 grams of the liquid savoryflavor, for storage. The water activity of the solution was 0.953@24.7°C., the refractive index was 1.4328, and the final concentration of thissavory flavor was 0.10% of Compound 1. Analysis via HPLC showed theaverage recovery of Compound 1 was 94% of theoretical.

Pizza Sauce Preparation Procedures:

Two pizza sauce samples, one comprising 0.4% of MSG and one containingno MSG, but comprising 1.1 ppm of Compound 1 were prepared using theingredients listed in Tables 391 and 39-2 below, using the followingprocedure.

Salt, citric acid, sugar, the spices and herbs, including the MSG, werepre-blended as solids at 80-100 rpms in a 6 quart bowl and mixed using aKitchenAide blender for 5 minutes using a whip attachment. Water wasplaced in a clean kettle and stirred with a Lightnin mixer at 120-200rpms to create a vortex. The savory flavor form of Compound 1, 0.10%,was slowly added to the water and mixed for five minutes. The vinegarand solid spice mixture was slowly added with the mixer still blendingat 80-100 rpms until the dry seasonings were dissolved, approximately 5minutes. The tomato paste was added and the slurry was mixed anadditional 10. The resulting pizza sauce was transferred to a stainlesssteel six quart pot on a gas burner and was heated to pasteurizingtemperature (175-190° F.) and held for a minimum of two minutes, cooleddown to ambient (70+/−2° F.) temperature.

TABLE 39-1 Control Pizza sauce, with Monosodium Glutamate FormulaIngredient Grams % (wt/wt) Water 252.75 50.55 Tomato paste (retailRalphs 5TP1Q) 220.80 44.16 Salt (Morton Salt) 9.00 1.80 Sugar (C&H#801461) 7.95 1.59 Distilled vinegar 50 grain (Heinz HF6A06UW) 3.75 0.75Monosodium Glutamate (Ajinomoto 310704) 2.00 0.40 Black pepper 28 mesh(Pacific Spice SA#01406) 1.05 0.21 Onion powder (Pacific Spice SA#C1406)0.70 0.14 Garlic powder (American Ingredients GA45) 0.70 0.14 Citricacid (ADM S501132) 0.70 0.14 Ground oregano (McCormick 523561) 0.45 0.09Basil (McCormick 526961) 0.15 0.03 TOTAL 500 100 Analysis pH 3.92 % TA(Titratable Acidity as Acetic Acid) 0.72 Brix 16.72

TABLE 39-2 Pizza sauce, with 1.10 PPM of Compound 1, No MSG FormulaIngredient Grams % (wt/wt) Water 253.58 50.72 Tomato paste (retailRalphs 5TP1Q) 220.80 44.16 Salt (Morton Salt) 9.625 1.93 Sugar (C&H#801461) 7.95 1.59 Distilled vinegar 50 grain (Heinz) 3.75 0.75 Blackpepper 28 mesh (Pacific Spice SA#01406) 1.05 0.21 Onion powder (PacificSpice SA#C1406) 0.70 0.14 Garlic powder (American Ingredients BA45) 0.700.14 Citric acid (ADM S501132) 0.70 0.14 Concentrate comprising (0.10%Compound 1) 0.55 0.11 Ground oregano (McCormick 523561) 0.45 0.06 Basil(McCormick 526961) 0.15 0.03 TOTAL 500 100 Analysis ph 3.75 % TA 0.80Brix 16.10

Sensory Evaluation:

Ten trained tasters were trained to identify relative intensities of thefollowing taste attributes: salty, savory/meaty, umami, brothiness,bitter, linger and off flavors. A triangle difference test determines ifthere is a sensory difference between two products. Three blinded, codedand randomized samples are presented to the panelist. Two samples arethe same and one is different. To answer the test correctly, thepanelist must taste all three samples and correctly pick out the samplethey feel is different from the other two.

The samples were blind coded and presented in randomized order. Thepanelists each performed a total of two triangle tests, with 3 samplesper test

a. Test 1: 2 samples with 1.1 ppm Compound 1; one sample with MSG.

b. Test 2: 2 samples with MSG; one sample with 1.1 ppm Compound 1.

TABLE 39-3 Triangle test results, n = 20 (10 Panelists × 2 reps) SamplesTest 1 Test 2 Totals # Incorrect 7 4 11 # Correct 3 6 9 Total 10 10 20Significance 0.701 0.077 0.191 (p-value)

In test #1 only three of the ten panelists correctly identified thedifferent sample (i.e the sample comprising MSG). In test #2, six of theten panelists correctly identified the different sample (i.e. the samplecomprising Compound 1). From a total of twenty tests, nine testscorrectly identified the different sample.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1. A process of preparing a compound of Formula (V):

comprising the steps of: a) condensing R¹⁰R²⁰ NH or a salt thereof witha 2-chloro-2-oxoacetate ester having the formula:

in the presence of a tertiary amine base and a solvent or solventmixture comprising one or more of toluene, o-xylene, m-xylene, p-xylene,and nitrobenzene, to form a solution of a 2-oxoacetate ester having theformula:

wherein R is C₁-C₄ linear or branched alkyl; and b) reacting thesolution of the 2-oxoacetate ester formed in step (a) with R³⁰R⁴⁰NH orsalt thereof to form the compound of Formula (V); wherein R¹⁰ and R³⁰are each independently selected from the group consisting of optionallysubstituted arylalkyl, optionally substituted heteroarylalkyl, andoptionally substituted hetero-cycle alkyl; and R²⁰ and R⁴⁰ are eachindependently H or optionally substituted C1-C3 alkyl.
 2. The process ofclaim 1, wherein R¹⁰ and R³⁰ are independently optionally substitutedarylalkyl or optionally substituted heteroarylalkyl; wherein the aryl orheteroaryl moiety has 5 to 12 ring atoms; and the alkyl moiety is C₁-C₅alkylene or substituted C₁-C₅ alkylene.
 3. The process of claim 1,wherein R¹⁰ and R³⁰ are independently optionally substituted arylalkylor optionally substituted heteroarylalkyl; wherein the aryl orheteroaryl moiety is phenyl or pyridyl; and the alkyl moiety ismethylene or ethylene.
 4. The process of claim 1, wherein the compoundof Formula (V) is represented by Formula (Va):

wherien A and B are independently an aryl, heteroaryl, or heterocyclehaving 5 to 12 ring atoms; m and n are independently 0, 1, 2, or 3; R⁵⁰is hydrogen or an alkyl or substituted alkyl having 1 to 4 carbon atoms;R⁶⁰ is C₁-C₅ alkylene or substituted C₁-C₅ alkylene; R²⁰ and R⁴⁰ areeach independently H or optionally substituted C1-C3 alkyl; and R⁷⁰ andR⁸⁰ are each independently selected from the group consisting ofhydroxy, fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl,ethyl, isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy,and trifluoromethoxy.
 5. The process of claim 4, wherein the compound ofFormula (V) is represented by Formula (Va):

wherien A and B are independently phenyl or pyridyl; and R⁷⁰ and R⁸⁰ areeach independently selected from the group consisting of hydroxy,fluoro, chloro, NH₂, NHCH₃, N(CH₃)₂, CO₂CH₃, SEt, SCH₃, methyl, ethyl,isopropyl, vinyl, trifluoromethyl, methoxy, ethoxy, isopropoxy, andtrifluoromethoxy.
 6. The process of claim 1, wherein R is methyl.
 7. Theprocess of claim 1, wherein R is ethyl.
 8. The process of claim 1,wherein the solvent or solvent mixture for step (a) comprises toluene oro-xylene, m-xylene, p-xylene, or a mixture thereof.
 9. The process ofclaim 8, wherein the solvent or solvent mixture is toluene.
 10. Theprocess of claim 1, wherein the tertiary amine base is a trialkyl amine,a trialkoxyamine, or a heteroaromatic base comprising a pyridineresidue.
 11. The process of claim 10, wherein the tertiary amine base ischosen from triethylamine, pyridine, lutidine, and1,8-diazabicyclo[5.4.0]undec-7-ene.
 12. The process of claim 11, whereinthe tertiary amine base is triethylamine.
 13. The process of claim 1,wherein R¹⁰R²⁰NH or salt thereof in step (a) is initially present in theform of an ammonium salt.
 14. The process of claim 1, wherein thesolution of the 2-oxoacetate ester is treated by one or more additionalsteps of adding an aqueous solution of hydrochloric acid to the solutionof the 2-oxoacetate ester to form an organic liquid phase comprising the2-oxoacetate ester and an aqueous phase; isolating and drying theorganic liquid phase comprising the 2-oxoacetate ester to form a drysolution of the 2-oxoacetate ester.
 15. The process of claim 14, whereinthe process comprises the following steps: removing the organic solventsfrom the dry solution of the 2-oxoacetate ester to form a solid2-oxoacetate ester; and purifying the solid 2-oxoacetate ester.
 16. Theprocess of claim 1, wherein step (b) also comprises: mixing R³⁰R⁴⁰NH tothe solution of step (a) to form a reaction solution, and heating thereaction solution to form a compound of Formula (V).
 17. The process ofclaim 16, wherein step (b) further comprises cooling the reactionsolution to form a cooled solution of the compound of Formula (V);solidifying the compound of Formula (V) from the cooled solution byadding a dialkyl ether; and collecting the solid compound of Formula(V).
 18. The process of claim 17, wherein the process comprises thefollowing steps: treating the solid compound of Formula (V) obtained instep (b) with heptane to form a slurry of the compound of Formula (V);and collecting the compound of Formula (V) from the slurry to formpurified the compound of Formula (V).